WO2003086276A2 - Compositions et procedes pour administration genique a specificite de porte et traitement des infections - Google Patents

Compositions et procedes pour administration genique a specificite de porte et traitement des infections Download PDF

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WO2003086276A2
WO2003086276A2 PCT/US2003/010081 US0310081W WO03086276A2 WO 2003086276 A2 WO2003086276 A2 WO 2003086276A2 US 0310081 W US0310081 W US 0310081W WO 03086276 A2 WO03086276 A2 WO 03086276A2
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ligand
receptor
composition
cell
phage
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WO2003086276A3 (fr
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Robert Abbott
David Larocca
Andrew Baird
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Selective Genetics Inc
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Selective Genetics Inc
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1131—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/11—Antisense
    • C12N2310/111—Antisense spanning the whole gene, or a large part of it
    • C—CHEMISTRY; METALLURGY
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    • C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
    • C12N2760/00011—Details
    • C12N2760/12011—Bunyaviridae
    • C12N2760/12211—Phlebovirus, e.g. Rift Valley fever virus
    • C12N2760/12222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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    • C12N2795/00—Bacteriophages
    • C12N2795/00011—Details
    • C12N2795/14011—Details ssDNA Bacteriophages
    • C12N2795/14041—Use of virus, viral particle or viral elements as a vector
    • C12N2795/14043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2795/00—Bacteriophages
    • C12N2795/00011—Details
    • C12N2795/14011—Details ssDNA Bacteriophages
    • C12N2795/14041—Use of virus, viral particle or viral elements as a vector
    • C12N2795/14045—Special targeting system for viral vectors
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    • C12N2810/00—Vectors comprising a targeting moiety
    • C12N2810/50—Vectors comprising as targeting moiety peptide derived from defined protein
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    • C12N2810/00—Vectors comprising a targeting moiety
    • C12N2810/50—Vectors comprising as targeting moiety peptide derived from defined protein
    • C12N2810/80—Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates
    • C12N2810/85—Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates mammalian
    • C12N2810/851—Vectors comprising as targeting moiety peptide derived from defined protein from vertebrates mammalian from growth factors; from growth regulators

Definitions

  • This invention relates generally to non-target vectors with designed tropism used to deliver nucleic acids to infectable cells and to the selection of ligands that bind to cell surface receptors/moieties associated with infectious agent binding and intemalization.
  • Biowarfare and bioterrorism have been defined as the intentional or the alleged use of viruses, bacteria, fungi, parasites, or toxins to produce death or disease in humans, animals or plants.
  • viruses appear to pose the most significant threat of widespread harm, primarily due to their relative ease of both production and transmissibility, as well as a lack of medical treatments.
  • known viruses considered suitable as biowarfare agents include smallpox virus, and the hemorrhagic fever viruses, such as ebola virus, amongst others.
  • Smallpox virus or variola virus, is considered one of the most dangerous potential biological weapons.
  • Smallpox virus is the etiological agent responsible for smallpox, a viral disease unique to humans. Smallpox virus is transmitted from an infected person to a new victim by inhalation of air droplets or aerosols. Smallpox symptoms are typically first observed twelve to fourteen days following infection and include aching pains, fever and prostration. Several days later, a papular rash develops on the face and extremities. The rash soon becomes vesicular and pustular. Patients experience considerable pain due to the rash, and death usually occurs during the second week of infection. In five to ten percent of smallpox patients, more rapidly progressive, malignant disease develops, which is generally fatal within five to seven days.
  • Ebola virus belongs to a group of distinct families of viruses associated with viral hemorrhagic fever (NHF). Although some types of hemorrhagic fever viruses cause mild illnesses, many cause severe life-threatening diseases. A large number of viruses have been associated with VHF, including arenaviruses, bunyaviruses, filoviruses, and flaviviruses. Hemorrhagic fever viruses reside naturally in an animal host, typically rodents and arthropods. These viruses are transmitted to humans through contact with bodily excretions from infected animals or mosquito or tick bites. Some viruses that cause NHF can spread from one person to another, once an initial person has become infected.
  • NHF viral hemorrhagic fever
  • Such viruses include the Ebola, Marburg, Lassa, and Crimean-Congo hemorrhagic fever viruses. This type of secondary transmission of the virus can occur directly, through close contact with infected people or their body fluids. It can also occur indirectly, through contact with objects contaminated with infected body fluids.
  • Ebola hemorrhagic fever is a severe, often-fatal disease in humans and nonhuman primates that has appeared sporadically since it was first recognized in 1976.
  • the disease is caused by infection with Ebola virus, a member of a family of R ⁇ A viruses called the Filoviridae.
  • Ebola virus a member of a family of R ⁇ A viruses called the Filoviridae.
  • the fourth, Ebola-Reston causes disease in nonhuman primates.
  • Ebola patients Within several days of infection, Ebola patients typically develop fever, headache, muscle aches, stomach pain, fatigue and diarrhea. Within one week, patients generally experience shock and death. Other symptoms experienced by some Ebola patients include blindness and bleeding.
  • Such agents can almost certainly be developed at a faster pace than vaccine defenses against those agents.
  • Relevant examples from nature include influenza, which mutates rapidly enough to require a new vaccine every year.
  • Fourth, not all vaccine programs are successful due to molecular mimicry, lack of antigenicity, and other phenomena that mask viral agents from immune recognition.
  • Most current methods of creating peptide fragments of infectious pathogens for generating an immune response are primarily based upon antigenicity, and without foreknowledge of whether the immune response will be neutralizing of infection.
  • Vaccine programs for human immunodeficiency virus (HIN), herpes viruses, and hepatitis C virus, for example have yet to succeed despite enormous commitments of time and resources.
  • the present invention provides vectors, compositions, and methods for delivery of a therapeutic gene(s) to an infectable cell using non-mammalian viruses that express a ligand capable of directing intemalization via the cell surface receptor(s) associated with the infectivity of the pathogenic agent.
  • the present invention provides vectors and methods useful for identifying receptors for infectious agents and those receptors' cognate binding partners.
  • the invention also provides compositions and methods, including vaccines, useful for preventing infection, as well as other advantages.
  • the present invention encompasses platform technology related to antimicrobial compositions and delivery of these compositions to infected cells or cells at risk of or capable of being infected (i.e. infectable cells).
  • the invention provides anti-microbial compositions and kits comprising vectors designed to bind certain cells, such as filamentous phage engineered to bind a molecule or cell surface protein associated with microbial infection and an anti-microbial agent. Accordingly, the invention also provides a method of identifying cell surface proteins/moieties associated with microbial infection.
  • the invention provides methods of delivering the anti-microbial agents to cells and methods of preventing, treating, or slowing a microbial infection.
  • the present invention provides an anti-microbial composition, comprising a delivery vehicle, such as filamentous phage presenting a ligand on their surface, and a physiologically acceptable excipient or diluent.
  • a delivery vehicle such as filamentous phage presenting a ligand on their surface
  • a physiologically acceptable excipient or diluent such as Ml 3, fl , or fd.
  • the filamentous phage may contain a heterologous nucleic acid sequence that encodes an anti-microbial agent, and may also contain a nucleic acid encoding a ligand expressed on their surface that binds to a moiety or protein present on the surface of a cell, or the ligand may be covalently conjugated, bound, or adhered to the vector, thereby providing portal-level specificity.
  • the ligand binds to a cell surface receptor/moiety.
  • the cell surface receptor is a viral receptor.
  • the cell surface receptor is upregulated during microbial (e.g. viral) infection or is expressed in substantially the same cells as a viral receptor/moiety.
  • the invention may be targeted to any cell surface receptor/moiety associated with microbial infection.
  • the receptor is heparan sulfate, ⁇ v ⁇ (vitronectin receptor), Ncam-1, sialylated glycophorin A, Pvr, Icam-1, CD55, Car, ⁇ 2 ⁇ r.
  • integrin (Nla-2), sialic acids, HANCr-1, low-density lipoprotein receptor protein family, Bgp (biliary glycoprotein), aminopeptidase ⁇ , major histocompatiblity class I molecule, high-affinity laminin receptor, nicotinic acetycholine receptor, neural cell adhesion molecule CD56, low-affinity nerve growth factor receptor, membrane cofactor protein CD46, asialoglycoprotein receptor Gp-2, ⁇ -dystroglycan, CD4, galactosylceramide, Cxcr4, Glvrl, Ram-1, Car, Tva, BLNRcp 1, CxcR family of receptors, major histocompatibilty complex class II molecule, ⁇ m ⁇ 2 , ⁇ v integrins, complement receptor Cr2 (CD21), epidermal growth factor receptor, fibroblast growth factor receptor, folate receptor alpha, CCR4, CCR5, chemokine receptors, IL-2 receptor,
  • the invention also includes any ligand capable of binding to a moiety or cell surface receptor associated with microbial infection.
  • the ligand is a natural ligand for a cell surface receptor, while in other embodiments, the ligand is a synthetic ligand for a cell surface receptor.
  • the ligand is an antibody or fragment thereof that specifically binds to a cell surface receptor. In one embodiment, the antibody is a single-chain antibody.
  • the ligand may be coupled to the vector, such as phage, by any suitable mechanism.
  • the ligand is genetically fused with a filamentous phage surface protein, and in another embodiment, the ligand is chemically conjugated to a filamentous phage surface protein.
  • the ligand is bound to a filamentous phage surface protein through charge-charge interactions.
  • the charge-charge interactions are provided by ligand/anti-ligand binding interactions.
  • the surface protein is a capsid protein. In specific embodiments, the capsid protein is a product of gene III, gene VI, gene NIII, or gene IX.
  • the ligand contains an endosomal escape moiety, and in other embodiments, the ligand contains a nuclear localization sequence.
  • the phage display an endosomal escape peptide moiety on their surface, and in another embodiment, the phage display a nuclear localization sequence on their surface.
  • the anti-microbial agent is a polypeptide, a ribozyme, an intrabody, or an antisense oligonucleotide. In one embodiment, the anti-microbial agent specifically interacts with a microbe gene or microbe or microbe-induced polypeptide.
  • the microbe gene or polypeptide is reverse transcriptase, R ⁇ A polymerase, a protease, ahelicase, VP35, VP40, NP30, VP24, a leader sequence, an early gene, a late gene, thymidine kinase, a toxin, gpl20, a cytokine, a transcription regulator, a nucleic acid binding protein, an immunomodulator, a coat protein, a capsid protein, an envelope protein, or a metabolic regulator.
  • the anti-microbe agent specifically interacts with a cellular gene or polypeptide.
  • the cellular gene or polypeptide is poly(A) binding protein, a cytokine, a nucleic acid binding protein, an immunomodulator, a transcription regulator, or a metabolic regulator.
  • the anti-microbial agent is capable of enhancing immune responsiveness, while in another embodiment, the anti-microbial agent is capable of decreasing viral replication.
  • the anti-microbe agent is operably linked to and under the control of a constitutive promoter, while in another embodiment, the anti-microbe agent is operably linked to an under the control of an event specific promoter.
  • the event specific promoter is a tissue-specific promoter, and in certain embodiments, the event-specific promoter is active primarily in virally infected cells.
  • the ligand binds to a moiety or protein present on the surface of an endothelial cell, a fibroblast cell, a lymphocyte, a neuronal cell, a macrophage, ahepatocyte, akeratinocyte, lung, vaginal, intestinal, stomach, trachea, nasal, kidney, hepatic, phogocyte, ovarian, or testes epithelial cells, a myocyte, a myoblast, hematopoietic cells, a bone marrow cell, or a stem cell.
  • the anti-microbial agent is active against one or more viruses. In certain embodiments, the anti-microbial agent is specific for one or more of Variola, Ebola, Marburg, Reston, Junin, Lassa, Crimean-Congo, Hanta, Epstein-Barr,
  • Venezuelan equine encephalitis, West Nile, and Rift Valley Fever viruses Venezuelan equine encephalitis, West Nile, and Rift Valley Fever viruses.
  • the virus is associated with hemmorhagic fever.
  • the invention provides a method for the delivery of an anti-microbial agent, comprising contacting a cell with an anti-microbial agent of the invention.
  • the invention provides a vector, such as a filamentous phage particle presenting a ligand on its surface, wherein the phage genome encodes a gene product under control of a promoter for use in treating microbial infection.
  • the invention provides a method for treating or slowing a microbial infection, comprising contacting a cell with an anti-microbial composition or filamentous phage particle of the invention.
  • the invention provides a kit comprising a container, an anti-microbial composition or filamentous phage particle of the invention, and instructions for use of said anti-microbial composition.
  • the invention provides a method of identifying a microbial epitope involved in host cell attachment or intemalization, involving contacting one or more ligand displaying genetic package with a cell(s), wherein the ligand comprises a microbe epitope, wherein the package comprises a nucleic acid encoding a detectable product that is expressed upon intemalization of the package, and wherein the cell(s) is capable of being infected by a microbe, followed by detecting product expressed by the cell(s) and recovering a nucleic acid molecule encoding a microbe epitope from the cell(s), thereby identifying a microbe epitope involved in host cell attachment or intemalization.
  • the ligand displaying genetic packages comprise or constitute a library of ligand displaying packages.
  • the library may be a cDNA library, such as a vims cDNA library.
  • the vims is Variola, Ebola, Marburg, Reston, Junin, Lassa, Crimean-Congo, Hanta, Epstein-Barr, Venezuelan equine encephalitis, West Nile, or Rift Valley Fever virus.
  • the detectable product is green fluorescent protein, ⁇ -galactosidase, secreted alkaline phosphate, chloramphenicol acetyltransferase, luciferase, human growth hormone or neomycin phosphotransferase.
  • the invention provides a method of preventing microbial infection, comprising delivering a microbe epitope identified according to a method of the invention to a patient.
  • the epitope is linked to a phage.
  • the invention provides a composition comprising a viral epitope identified according to a method of the invention.
  • the epitope is linked to a phage.
  • the invention provides a method of inducing an immune response in a patient, comprising delivering a microbe epitope identified according to a method of the invention to a patient.
  • the epitope is linked to a phage.
  • the compositions of the invention further comprise a physiologically acceptable carrier.
  • the present invention provides platform technology useful for treating a variety of intracellular infections, particularly those currently associated with high mortality or incapacity, such as biowarfare agents.
  • This technology contemplates delivering nucleic acids to infectable cells using non-target vectors, such as non- mammalian viruses, designed to target infectable cells.
  • non-target vectors such as non- mammalian viruses
  • infectious cells include any cell infected, at risk of infection, or capable of being infected by a microbe of interest.
  • the technology provided by the invention offers a variety of advantages, including the ability to rapidly engineer the platform to target different infectious agents/microbes and deliver different nucleic acids to such cells.
  • the platform technology of the invention contemplates four principle concepts that may conveniently be described as recognition, expression, payload, and distribution.
  • Recognition refers to the identification of the ligand/receptor pairing of a given infectious agent, such as a biowarfare agent, and the identification of a peptide that imparts similar intemalization qualities and specificity as the pathogen.
  • Expression includes vector characteristics and parameters associated with protein or mRNA expression from the delivered genes.
  • Payload describes the molecules being delivered to infectable cells and contemplates a wide variety of molecules, such as polynucleotides, including therapeutic polynucleotides, for example.
  • Distribution refers to the pharmaceutical and physiological characteristics of the compounds and compositions of the invention, and methods of introducing the compounds and compositions to a target cell.
  • the present invention is generally directed to constructs and compositions suitable for treatment of intracellular infections and methods of specifically delivering such compositions to infectable cells.
  • One application of the present invention utilizes a non- mammalian virus displaying a ligand of a cell surface receptor associated with infection to deliver therapeutic compositions to infectable cells.
  • Another application of the invention involves using a non-target vector to identify a cognate binding partner to a cell surface receptor associated with infection. While primarily relevant to viral pathogens, these compounds, compositions and methods should be useful against any intracellular pathogen or infectious agent that internalizes through receptor-based mechanisms.
  • the term "virus” or "viral pathogen” will also refer to any intracellular microbe that infects cells primarily by way of a receptor(s)-based or cell surface moiety recognition and intemalization mechanism.
  • the invention includes a delivery vehicle/non-target vector that provides a genetic payload to an infectable cell via the same or similar cell surface receptor(s) or cell surface moiety used by the infectious agent to gain access to the cell.
  • the delivery vehicle comprises a ligand or binding partner for such cell surface moiety utilized by the infectious agent.
  • ligand refers to any peptide, polypeptide, protein or non-protein, such as a peptidomimetic, that is capable of binding to such cell surface moiety and internalizing.
  • the cell surface molecule is the microbe receptor.
  • the "viral receptor” is any known or unknown cell surface moiety (e.g.
  • bind to a receptor refers to the ability of a ligand to specifically recognize and detectably bind to a receptor or combination of receptors, as assayed by standard in vitro assays.
  • a molecule is said to "specifically bind" to a particular peptide or polypeptide if it binds at a detectable level with the particular peptide polypeptide, but does not bind detectably with another polypeptide containing an unrelated sequence.
  • a prototype phage vector system that can deliver and express genes in mammalian cells by utilizing cognate ligand/receptor interactions for intemalization was recently described in U.S. Patent No. 6,054,312. Specifically, filamentous phage particles presenting a mutated fibroblast growth factor (FGF) on their surfaces were used to deliver green fluorescent protein (GFP) encoded by the phage genome to a mammalian cell expressing FGF receptors on its surface.
  • FGF mutated fibroblast growth factor
  • GFP green fluorescent protein
  • filamentous phage were discounted as suitable for this purpose because there are no naturally occurring receptors on mammalian cells for phage entry and because they are single-stranded DNA viruses and do not contain promoters that operate in mammalian cells.
  • bacteriophage offers distinct advantages over mammalian viruses regarding their use as gene therapy vectors.
  • Bacteriophage provide a natural method of condensing and packaging therapeutic DNA, and they have no known pathogenicity or toxicity in mammals.
  • bacteriophage are comparatively simple entities, large scale production is easier and less expensive than the production of mammalian viral vectors.
  • bacteriophage can be "evolved" easily to produce attractive pharmaceutical characteristics (i.e., long half-lives in blood, reduced immune system recognition, oral availability, and high stability).
  • Delivery vehicles refer to any non-target vectors, such as a non-mammalian vims that comprises a peptide/protein ligand and is capable of being internalized into a target cell.
  • Non-target vector refers to a vector that either naturally has no native tropism for a target cell or has been modified such that the natural tropism has been ablated or removed.
  • Target cells include any cell that is not normally transfected or infected by a specific delivery vehicle. Thus, target cells may include mammalian cells and plant cells, for example. Typically, a target cell is infected by, or at risk of being infected by, an infectious agent, such as a virus.
  • intemalization into the target cell is preferably mediated by a peptide/protein ligand displayed on the surface of the delivery vehicle.
  • Delivery vehicles are typically used to deliver a nucleic acid molecule to a target cell. Delivery vehicles comprising such "genetic payloads" are herein referred to as "ligand displaying genetic packages” or “ligand-bearing non-target vectors.”
  • a "genetic payload” includes any nucleic acid molecule itself capable of detection, or expressing a molecule capable of detection, once internalized in a target cell.
  • ligand displaying genetic packages may be used within the context of the present invention.
  • a nucleic acid carried by the ligand displaying genetic package is expressed upon intemalization into the cell, thereby allowing for recovery and detection of an internalized genetic payload.
  • the expressed nucleic acids provide a therapeutic effect to a target cell or patient.
  • the ligand displaying genetic package may carry a nucleic acid molecule which allows for detection via PCR of unique sequences, the Hirt extraction method, or by the ability of the internalized nucleic acid sequences to bind non-endogenous DNA binding proteins (e.g., nucleic acid sequence could comprise a lac operon, thereby allowing for lac repressor binding).
  • display may be by a viras, RNA-peptide fusions, bacteriophage, bacteria, or similar system (See, Phage Display of Peptides and Proteins, Kay (Ed.), Academic Press, San Diego, pp. 151-193, 1996).
  • a variety of delivery vehicles are contemplated by the current invention. Although most human gene therapy viral vectors described thus far are mammalian viruses possessing the ability to infect mammalian cells, preferred delivery vehicles of the present invention do not normally bind to or internalize within target cells. Generally, a non-target virus of the invention has no natural tropism for the organism to be treated. Thus, non- mammalian viruses are preferred delivery vehicles for mammalian target cells. Similarly, non-plant viruses are preferred delivery vehicles for plant target cells. Because the delivery vehicles of the present invention are normally incapable of infecting or transducing target cells, they can be engineered to bind and internalize within specific target cells. Thus, the present invention avoids previous problems with target-based systems (e.g.
  • non- mammalian virus means non-target viras.
  • non-target viruses included mammalian viruses used to target non-mammalian cells. Examples of specific non-mammalian viruses that may be used according to the present invention include baculovirus, tobacco mosaic virus (TMV), tomato spotted wilt virus (SWV), tobacco etch vims (TEV), tobacco necrosis vims (TNV), wheat streak mosaic viras (WSMV), soil bome wheat mosaic virus (SBWMV), barley yellow dwarf virus (BYDN), and bacteriophage.
  • TMV tobacco mosaic virus
  • SWV tomato spotted wilt virus
  • TMV tobacco etch vims
  • TMV tobacco necrosis vims
  • WSMV wheat streak mosaic viras
  • SBWMV soil bome wheat mosaic virus
  • BYDN barley yellow dwarf virus
  • bacteriophage include the filamentous phages, lambda, T4, MS2, and the like.
  • bacteriophage of the invention include, for example, Tl, T2, T3, T4, T5, T6, T7, SPO1, Ff (filamentous), MS2, ⁇ 29, ⁇ X174, lambdoid, P22, P2, P4,P1,N4, PBS1, PBS2, PM2, and Mu.
  • a preferred phage is a filamentous phage, such as Ml 3, fl, or fd, described below. Accordingly, many illustrations, while exemplifying the use of phage, could also be performed with any ligand displaying genetic package.
  • Filamentous phage encompasses a group of bacteriophages that are able to infect a variety of Gram-negative bacteria through interaction with the tip of the F pilus.
  • Well known filamentous phages include Ml 3 , fl , and fd.
  • the genomes of these phage are single-stranded DNA, but replicate through a double-stranded form. Phage particles are assembled in the bacteria and extraded into the media. Because the bacteria continue to grow and divide, albeit at a slower rate than uninfected cells, relatively high titers of phage are obtained. Moreover, replication and assembly appear to be unaffected by the size of the genome.
  • filamentous phage are a valuable tool in molecular biology.
  • Further development of filamentous phage systems have led to the production of cloning vectors, called phagemids, that combine features of plasmids and phages.
  • Phagemids contain an origin of replication and packaging signal of the filamentous phage, as well as a plasmid origin of replication.
  • Other elements that are useful for cloning and/or expression of foreign nucleic acid molecules are generally also present. Such elements include, without limitation, selectable genes, multiple cloning site, primer sequences.
  • filamentous phage particles refers to particles containing either a phage genome or a phagemid genome. The particles may contain other molecules in addition to filamentous capsid proteins.
  • Filamentous phages have also been developed as a system for displaying proteins and peptides on the surface of the phage particle.
  • fusion proteins are produced that are assembled into the capsid (Smith, Science 228: 1315, 1985; U.S. Patent No.5,223,409).
  • the foreign protein or peptide is displayed on the surface of the phage particle.
  • Methods and techniques for phage display are well known in the art (see also, Kay et al, Phage Display of Peptides and Proteins: A Laboratory Manual, Academic Press, San Diego, 1996). While the current invention provides for the use of any non-mammalian viral vector, including a variety of phage vectors, filamentous phage is utilized as an exemplary vector throughout. 3.
  • Vectors include a variety of phage vectors, filamentous phage is utilized as an exemplary vector throughout. 3.
  • Delivery vehicles and ligand displaying genetic packages are produced using vectors or may themselves be non-target vectors.
  • a "vector" includes any nucleic acid molecule, compound, or composition that may be used to carry and/or replicate a heterologous polynucleotide.
  • Typical vectors include plasmids, cosmids, viruses, phages, liposomes, particles (e.g. polymer particles), and yeast artificial chromosomes (YACs), for example.
  • YACs yeast artificial chromosomes
  • genetic payloads are inserted into vectors.
  • Vectors utilized in the present invention include, but are not limited to, any non-mammalian viral vector. Non-mammalian viral vectors are widely available commercially and within the scientific community.
  • Preferred vectors are filamentous phage vectors.
  • Filamentous phage offer numerous advantages as vectors. Filamentous phage possess a lower complexity genome than most other non-mammalian viruses, which allows easier genetic manipulation. This, along with other characteristics, permits more rapid design and production of vectors specifically targeting specific infections.
  • Phage coat proteins can display viral receptors and ligands. For example, the adenoviral penton base protein was displayed on phage (DiGiovine, M., et al. Virology 282:102-12 (2001)). In the context of biowarfare, such ease of design and simplicity of manufacturing may prove critical for launching rapid and successful countermeasures, particularly against newly engineered viral and microbial weapons. In addition, these characteristics provide a flexible and easily altered platform from which to rapidly develop vectors modified to target specific cells, provide specific therapeutic molecules, or fight specific viral infections.
  • Filamentous phage vectors generally fall into two categories : phage genome and phagemids. Either type of vector may be used within the context of the present invention, although phage genome vectors are utilized preferentially. Many such commercial vectors are available. For example, the pEGFP vector series (Clontech; Palo Alto, CA), M13mp vectors (Pharmacia Biotech, Sweden), pCANTAB 5E (Pharmacia Biotech), pBluescript series (Stratagene Cloning Systems, La Jolla, CA), and others may be used.
  • the vector must accept a cassette containing a promoter and an exogenous polynucleotide sequence (transgene) in operative linkage. Any promoter that is active in the cells to be transfected can be used.
  • the vector must also have a phage origin of replication and DNA sequences needed for phage particle assembly.
  • the construct includes a transcription terminator sequence, including a polyadenylation sequence, splice donor and acceptor sites, and an enhancer.
  • a transcription terminator sequence including a polyadenylation sequence, splice donor and acceptor sites, and an enhancer.
  • Other elements useful for expression and maintenance of the construct in mammalian cells or other eukaryotic cells may also be incorporated (e.g., origin of replication).
  • vectors include an SN40 origin of replication to enhance gene expression by allowing replication of the plasmid to high copy number in cells that make SN40 T antigen. Because the vector constructs are conveniently produced in bacterial cells, elements that are necessary or enhance propagation in bacteria may be incorporated. Such elements include an origin of replication, selectable marker and the like (see discussion below).
  • the vector may contain a transgene whose product can be detected or selected for.
  • a reporter gene is one whose product can be detected, such as by fluorescence, enzyme activity on a chromogenic or fluorescent substrate, and the like or selected for by growth conditions.
  • reporter genes include, without limitation, green fluorescent protein (GFP), ⁇ -galactosidase, chloramphenicol acetyltransferase (CAT), luciferase, neomycin phosphotransferase, secreted alkaline phosphatase (SEAP), and human growth homione (HGH).
  • Selectable markers include drag resistances, such as neomycin (G418), hygromycin, and the like.
  • the promoter that controls expression of the transgene should be active or activatable in the targeted cell.
  • the targeted cell may be mammalian, avian, plant, and the like. Most applications of the present invention will involve transfection of mammalian cells, including human, simian, canine, feline, equine, bovine, and the like.
  • the choice of the promoter will depend in part upon the targeted cell type and the degree or type of control desired.
  • Promoters that are suitable within the context of the present invention include, without limitation, constitutive, inducible, tissue specific, cell type specific, temporal specific, or event-specific. Examples of constitutive or nonspecific promoters include the S V40 early promoter (U.S. Patent No.
  • SV40 late promoter U.S. PatentNo. 5,118,627)
  • CMN early gene promoter U.S. PatentNo. 5,168,062
  • bovine papilloma virus promoter bovine papilloma virus promoter
  • adenovirus promoter adenovirus promoter
  • cellular promoters are also amenable within the context of this invention.
  • cellular promoters for the so- called housekeeping genes are useful (e.g., ⁇ -actin).
  • Viral promoters are generally stronger promoters than cellular promoters. Inducible promoters may also be used.
  • promoters include MMTN LTR (PCT WO 91/13160), inducible by dexamethasone, metallothionein, inducible by heavy metals, and promoters with cAMP response elements, inducible by cAMP, heat shock, promoter.
  • MMTN LTR PCT WO 91/13160
  • inducible by dexamethasone PCT WO 91/13160
  • metallothionein inducible by heavy metals
  • promoters with cAMP response elements inducible by cAMP, heat shock, promoter.
  • Event-type specific promoters are active or upregulated only upon the occurrence of an event, such as tumorigenicity or viral infection, for example.
  • the HIN LTR is a well-known example of an event-specific promoter.
  • the promoter is inactive unless the tat gene product is present, which occurs upon viral infection.
  • Some event-type promoters are also tissue-specific.
  • Preferred event-type specific promoters include promoters activated upon viral infection.
  • the serum amyloid A activating factor is an additional example of a transcription factor that binds and activates S AF-regulated genes in an event-type manner.
  • SAF was identified as a family of inducible transcription factors that is activated by many mediators of inflammation. Ray, A. et al, DNA Cell Biol. 21(l):31-40, 2002.
  • promoters discussed herein include, but are not limited to, promoters for alphafetoprotein, alpha actin, myo D, carcinoembryonic antigen, NEGF- receptor (GenBank Accession No. X89776); FGF receptor; TEK or tie 2 (GenBank Accession No. L06139); tie (GenBank Accession Nos. X60954; S89716); urokinase receptor (GenBank Accession No. S78532); E- and P-selectins (GenBank Accession Nos. M64485; L01874); NCAM-1 (GenBank Accession No. M92431); endoglin (GenBank Accession No.
  • HUMCD44B CD40 (GenBank Accession Nos. HACD40L; HSCD405FR); vascular-endothelial cadherin (Martin-Padura et al, J. Pathol 775:51, 1995); notch 4 (Uyttendaele et al, Development 122:2251, 1996) high molecular weight melanoma-associated antigen; prostate specific antigen- 1, probasin, FGF receptor, NEGF receptor, erb B2; erb B3; erb B4; MUC-1; HSP-27; int-1; int-2, CEA, HBEGF receptor; EGF receptor; tyrosinase, MAGE, IL-2, IL-2 receptor; prostatic acid phosphatase, probasin, prostate specific membrane antigen, alpha-crystallin, EGFR, PDGF receptor, integrin receptor, ⁇ -actin, SMI and SM2 myosin heavy chains, calponin-
  • repressor sequences may be inserted to reduce non-specific expression of the polynucleotide or gene comprising the genetic payload.
  • Multiple repressor elements may be inserted in the promoter region. Repression of transcription is independent of the orientation of repressor elements or distance from the promoter.
  • One type of repressor sequence is an insulator sequence.
  • Negative regulatory elements have been characterized in the promoter regions of a number of different genes.
  • the repressor element functions as a repressor of transcription in the absence of factors, such as steroids, as does the NSE in the promoter region of the ovalbumin gene (Haecker et al, Mol. Endocrinology 9:1113-1126, 1995).
  • These negative regulatory elements bind specific protein complexes from oviduct, none of which are sensitive to steroids.
  • Three different elements are located in the promoter of the ovalbumin gene. Oligonucleotides corresponding to portions of these elements repress viral transcription of the TK reporter.
  • One of the silencer elements shares sequence identity with silencers in other genes (TCTCTCCNA).
  • Repressor elements have also been identified in the promoter region of a variety of genes, including the collagen II gene, for example. Gel retardation studies showed that nuclear factors from HeLa cells bind specifically to DNA fragments containing the silencer region, whereas chondrocyte nuclear extracts did not show any binding activity (Savanger et al, J. Biol. Chem. 265( ⁇ 2):6669-6674, 1990). Repressor elements have also been shown to regulate transcription in the carbamyl phosphate synthetase gene (Goping et al, Nucleic Acid Research 23(10):1717-1721, 1995). This gene is expressed in only two different cell types, hepatocytes and epithelial cells of the intestinal mucosa.
  • Negative regulatory regions have also been identified in the promoter region of the choline acetyltransferase gene, the albumin promoter (Hu et al, J. Cell Growth Differ. 3(9):577-588, 1992), phosphoglycerate kinase (PGK-2) gene promoter (Misuno etal, Gene 119(2):293-297 ' , 1992), and in the 6-phosphofructo-2-kinase/fractose- 2, 6-bisphosphatase gene, in which the negative regulatory element inhibits transcription in non-hepatic cell lines (Lemaigre etal, Mol. Cell Biol. 77(2):1099-1106).
  • Tse-1 has been identified in a number of liver specific genes, including tyrosine aminotransferase (TAT). TAT gene expression is liver specific and inducible by both glucocorticoids and the cAMP signaling pathway.
  • the cAMP response element (CRE) has been shown to be the target for repression by Tse-1 and hepatocyte- specific elements (Boshart etal, Cell 67(5):905-916, 1990). Accordingly, it is clear that a variety of such elements are known or are readily identified.
  • elements that increase the expression of the desired product are incorporated into the construct.
  • Such elements include internal ribosome binding sites (IRES; Wang and Siddiqui, Curr. Top. Microbiol Immunol 203:99, 1995; Ehrenfeld and Semler, Curr. Top. Microbiol Immunol. 203:65, 1995; Rees et al., Biotechniques 20:102, 1996; Sugimoto et al., Biotechnology 12:694, 1994).
  • IRES increase translation efficiency.
  • other sequences may enhance expression. For some genes, sequences especially at the 5' end inhibit transcription and/or translation. These sequences are usually palindromes that can form hairpin structures. Any such sequences in the nucleic acid to be delivered are generally deleted.
  • Transcript levels may be assayed by any known method, including Northern blot hybridization, Rnase probe protection and the like. Protein levels may be assayed by any known method, including ELISA.
  • the phage has an origin of replication suitable for the target cells.
  • Viral replication systems such as EBV ori and EBNA gene, SV40 ori and T antigen, or BPN ori, may be used.
  • Other mammalian replication systems may be interchanged.
  • the replication genes may cause high copy number. Expression of therapeutic genes from the phage genome may be enhanced by increasing the copy number of the phage genome.
  • the SV40 origin of replication is used in the presence of SN40 T antigen to cause several hundred thousand copy number.
  • the T antigen gene may be already present in the cells, introduced separately, or included in the phage genome under the transcriptional control of a suitable cell promoter.
  • Other viral replication systems for increasing copy number can also be used, such as EBV origin and EB ⁇ A.
  • peptides or other moieties that allow or promote the escape of the vectors (and any molecule attached thereto or enclosed therein) from the endosome are incorporated and expressed on the surface of the bacteriophage.
  • Such "other moieties” include molecules that are not themselves peptides but which have the ability to disrupt the endosomal membrane, thereby facilitating the escape of the vector, and molecules that otherwise mimic the endosomal escape properties of the within-described peptide sequences (see, e.g., published PCT Publication No. WO96/10038 and Wagner et al., PNAS S9:7934-7938, 1992, the disclosures of which are incorporated by reference herein).
  • Peptide sequences that confer the ability to escape the endosome are particularly preferred. Such sequences are well known and can be readily fused covalently or genetically to a coat protein, such as gene III or gene VIII of filamentous phage. Although fusion of one or more peptide sequences to a coat protein is described herein as a preferred embodiment, it should be understood that other methods of attachment and other moieties besides peptide are useful as disclosed herein.
  • an example of a dual display filamentous phage presents a ligand as a fusion to gene III and an endosomal escape peptide fused to gene VIII.
  • the locations of the ligand and escape sequences are interchangeable and furthermore may reside in the same fusion protein.
  • Escape sequences that are suitable include, without limitation, the following exemplary sequences: a peptide of Pseudomonas exotoxin (Donnelly, J.J., et al., PNAS, 90, 3530-3534, 1993); influenza peptides such as the HA peptide and peptides derived therefrom, such as peptide FPI3; Sendai Virus fusogenic peptide; the fusogenic sequence from HIV gpl protein; Paradaxin fusogenic peptide; and Melittin fusogenic peptide (see published PCT Publication No.
  • a peptide of Pseudomonas exotoxin Donnelly, J.J., et al., PNAS, 90, 3530-3534, 1993
  • influenza peptides such as the HA peptide and peptides derived therefrom, such as peptide FPI3
  • Sendai Virus fusogenic peptide the fusogenic sequence from HIV
  • GLFEAIEGFIENGWEGMIDGGGC SEQ ID NO: 1
  • fusogenic peptides examples of two other endosome-disraptive peptides, sometimes called fusogenic peptides, are: GLFEAIEGFIENGWEGMIDGGGC (SEQ ID NO: 1); and
  • GLFEAIEGFIENGWEGMIDGWYGC SEQ ID NO: 2.
  • Other peptides useful for disrupting endosomes may be identified by various general characteristics. For example, endosome-disrupting peptides are often about 25-30 residues in length, contain an alternating pattern of hydrophobic domains and acidic domains, and at low pH (e.g. , pH 5) form amphipathic ⁇ -helices. The endosome-disraptive peptide may be present as single or multiple copies at the N- or C- terminus of the ligand.
  • Escape peptides may also be selected using a molecular evolution strategy.
  • a library of random peptides is engineered into the gene VIII protein of a phage vector that has a ligand fused to gene III and that carries a detectable (e.g., GFP) or selectable marker (e.g., drug resistance) .
  • Mammalian cells are infected with the library and the cells selected by detection of the marker. The cells that have the most efficient endosomal escape sequence should have the highest expression or most resistance. Multiple rounds of selection may be performed to reduce the complexity of the recovered peptide encoding genes.
  • the peptide genes are recovered and engineered into the phage vectors.
  • membrane-disruptive peptides may be incorporated into the complexes.
  • adenovirases are known to enhance disruption of endosomes.
  • Nims-free viral proteins such as influenza viras hemagglutinin HA-2, also disrupt endosomes and are useful in the present invention.
  • Other proteins may be tested in the assays described herein to find specific endosome disrupting agents that enhance gene delivery. In general, these proteins and peptides are amphipathic (see Wagner et al., _4 ⁇ -V. Drug. Del. Rev. 74:113-135, 1994).
  • Another sequence that may be included in a vector is a sequence that facilitates trafficking proteins into the nucleus.
  • the therapeutic nucleotide sequence is trafficked to the nucleus.
  • These so-called nuclear translocation or nuclear localization sequences ( ⁇ LS) are generally rich in positively charged amino acids. Because the carboxyl terminus of gene VIII protein of filamentous phage already carries a positive charge, increased charge and likeliness of nuclear transport may be enhanced by fusing known mammalian cell ⁇ LS sequences to the gene VIII protein. ⁇ LS fusions to other coat proteins of filamentous phage may be substituted. The ⁇ LS may be fused to a capsid protein distinct from a ligand/capsid fusion.
  • ⁇ LS sequences include those resembling the short basic ⁇ LS of the SV40 T antigen; the bipartite ⁇ LS of nucleoplasmin; the ribonucleoprotein sequence
  • ⁇ LS sequences include the HIV matrix protein ⁇ LS; and the nuclear translocation components importain/hSRP 1 and Ran/TC4; the consensus sequence KXX(K/R) (SEQ ID NO: 3) flanked by Pro or Ala; the nuclear translocation sequence of nucleoplasmin; or the NLS from antennapedia (See, WO 96/41606).
  • nucleic acid condensing peptides are linked to non-basic nuclear localization sequences which function to transport nucleoprotein.
  • sequences include influenza nucleoprotein and HIV MA protein.
  • Other useful NLS include hnRNP Al protein, a protein which transports ribonucleoprotein complexes.
  • Another useful NLS is included in peptides such as NBC 1 and NBC2, which also function to condense nucleic acid. (See, WO 96/41606)
  • the present invention thus contemplates use of the foregoing sequences as well as the others disclosed herein.
  • Additional factors that enhance expression of the transgene may be included. Such factors may be identified by a method in which sequences are fused to phage coat proteins and the phage are selected on the basis of efficient reporter gene expression.
  • Known DNA repair enzymes or polymerases from mammalian cells or single stranded DNA viruses are candidate sequences. Phage that present the ligand as a fusion with a phage coat protein are engineered to contain the appropriate coding regions. For the filamentous phages, usually genes III and VIII are used. Other capsid proteins may be substituted. Techniques for inserting ligand coding sequence into a phage gene are well known (see e.g., Sambrook et al., supra; Ausubel et al., supra). In certain embodiments, propagation or stable maintenance of the constmct may be desirable or may be necessary to attain a sufficient amount or concentration of the gene product for effective gene therapy. Examples of replicating and stable eukaryotic origins of replication are known.
  • the invention contemplates delivering polynucleotides to infectable cells with portal-level specificity.
  • portal-level specificity relates to the mechanism by which an infectious agent enters a cell.
  • the infectious agent is a virus that enters a cell via binding and internalizing through a cell surface receptor
  • the cell surface receptor is considered a portal of entry.
  • portal-level specificity means that the delivery vehicle is capable of binding a receptor involved in the process by which an infectious agent enters a cell. In other words, the delivery vehicle is capable of following an infectious agent into cells, typically via the same receptor the infectious agent used for entry.
  • Portal-level specificity is primarily achieved through the targeting ligand displayed on the surface of the vector of the invention.
  • Portal level specificity means the use of ligands capable of binding any of the following three groups of cell surface molecules: (1) receptors for infectious agents (e.g. viral receptors); (2) receptors that are upregulated during infection; and (3) receptors whose expression substantially correlates with or overlaps cells infected or capable of being infected by an infectious agent.
  • This third group of cell surface molecules includes cell surface molecules expressed on substantially the same cells as a viral receptor. Accordingly, all of these types of cell surface receptors are considered "infectious agent- associated receptors" or "virus-associated receptors", depending on the nature of the associated infectious agent.
  • a "receptor” includes any cell surface molecule involved in binding and/or intemalization into the cell.
  • receptors include molecules such as sugars and lipids, for example, in addition to polypeptides.
  • Virtual receptor refers to any portal of entry for a virus.
  • binding partner includes any molecule capable of binding to a specified molecule and, therefore, includes any and all of the three previously described groups of ligands, for example.
  • portal-level specificity is that it concentrates the genetic payload within infected or infectable cells, thus increasing potency, sheltering the compound from clearance mechanisms, and decreasing exposure of other cells and tissues, thereby lessening required dosage levels and risks of toxicity.
  • ligand/receptor pairing is a central process in viral infection and replication. Most intracellular pathogens gain entry into target cells through cell surface receptor interactions. These interactions are highly specific and often determine the tissue and species tropism of a pathogen.
  • a viral binding ligand and its target receptor becomes even more important because it defines the identity of a viras.
  • the ligand/receptor pairing determines what host species a virus infects, as well as what cell-type within that organism can be infected. To change the pairing is to change the viras.
  • Viral biowarfare agents are selected, in part, because of their pathogenic profile, a significant aspect of which is governed by the ligand/receptor tropism of the virus.
  • a platform for treatment based upon portal-level specificity presents numerous advantages, particularly in the context of biowarfare agents.
  • a platform can be readily altered (e. g. by changing the ligand) to target different infectious agents, without necessarily changing other characteristics of the delivery vehicle, such as the genetic payload, for example.
  • the genetic payload can be quickly adapted to combat different infectious agents or as a countermeasure against likely designer modifications, without changing the tropism or many of the underlying pharmacokinetic characteristics of the vehicle (i.e., changes in the genetic payload would be internal to the vehicle).
  • the invention includes vehicles and vectors that display single ligands, as well as vectors displaying multiple different ligands and vectors displaying multiple copies of ligands.
  • a vector permits multimeric display of three to five copies of a ligand. Multimeric display vectors are preferred for targeting receptors that require dimer formation for activation and/or intemalization.
  • ligands of the invention include any ligand capable of binding to a receptor associated with infection by any infectious agent.
  • an "infectious agent” is any living organism capable of infecting a host's cells. Infectious agents include, for example, bacteria, viruses, fungi, and protozoa. Examples of intracellular bacteria include, but are not limited to, Legionella, Chlamydia trachomatis, Brucella, Mycobacteria, Salmonella, and Listeria.
  • infectious viral agents include, but are not limited to, Adenovirus (types 1, 2, 3, 4, 5 and 7), Adenovirus (types 40 and 41), Bluetongue viras, Colorado tick fever viras, Crimean-Congo hemorrhagic fever virus, Cytomegalovirus, Dengue viras (1, 2, 3, 4), Eastern (Western) equine encephalitis viras, Ebola vims, Echovirus, Enterovirus 70, Epstein-Barr viras, Hantaviras, Hepatitis A virus, Hepatitis B vims, Hepatitis C virus, Hepatitis D viras, Hepatitis E viras, Herpes simplex viras, Herpesvirus simiae, Human coronaviras, Human immunodeficiency virus, Human papillomavirus, Human rotaviras, Human T-lymphotrophic viras, Influenza virus, Junin
  • infectious agents associated with human malignancies include Epstein-Barr viras, Helicobacter pylori, Hepatitis B virus, Hepatitis C virus, Human heresvirus-8, Human immunodeficiency virus, Human papillomaviras, Human T cell leukemia vims, liver flukes, and Schistosoma haematobium.
  • viruses are associated with viral hemorrhagic fever, including filovirases (e.g., Ebola, Marburg, and Reston), arenavirases (e.g. Lassa, Junin, and Machupo), and bunyaviruses.
  • filovirases e.g., Ebola, Marburg, and Reston
  • arenavirases e.g. Lassa, Junin, and Machupo
  • bunyaviruses e.g., phlebovirases, including, for example, Rift Valley fever viras, have been identified as etiologic agents of viral hemorrhagic fever.
  • Etiological agents of hemorrhagic fever and associated inflammation may also include paramyxovirases, particularly respiratory syncytial virus (Feldmann, H. etal. (1993) Arch Virol Suppl 7:81 -100).
  • togavirus Chikungunya
  • flaviviras dengue, yellow fever, Kyasanur Forest disease, Omsk hemorrhagic fever
  • nairoviras Crimian-Congo hemorrhagic fever
  • hantavims Hemorrhagic fever with renal syndrome, nephropathic epidemia.
  • SinNombre virus was identified as the etiologic agent of the 1993 outbreak of hantavims pulmonary syndrome in the American Southwest.
  • Vcam vascular cell adhesion molecule, Prrl, Prr2, PVr-related proteins 1 and 2
  • SCR short consensus repeat
  • Ig immunoglobulin
  • Tnf tumor necrosis factor
  • Carl cytopathic avian leucosis and sarcoma virus receptor
  • Car coxsackievirus and adenovirus receptor
  • HveA herpesvirus entry mediator.
  • the invention contemplates numerous modes of intemalization of ligand displaying genetic packages, including transformation, infection, transduction, conjugation, protoplast fusion, and transfection, for example.
  • Typical modes of viral entry include direct membrane fusion, endocytosis, and pH dependent entry, for example.
  • the ligand could be conjugated to a protein of a delivery vehicle such as a bacteriophage, either as a fusion protein, through chemical conjugation, or charge-charge interactions, and is used to deliver a genetic payload to a cell.
  • a protein of a delivery vehicle such as a bacteriophage
  • Numerous molecules are known that bind a specific receptor and are internalized, generally by way of endosomes. Yet other molecules are antibodies and antibody derivatives, both of which are also included within the definition of ligand.
  • methods are provided for the selection of additional ligands that satisfy the criteria presented above.
  • a "ligand/anti-ligand” pair refers to a complementary/anti-complementary set of molecules that demonstrate specific binding, generally of relatively high affinity.
  • Exemplary ligand/anti-ligand pairs include an antibody and its ligand as well as ligand/receptor binding. While it should be understood that the designation of either component of the above mentioned ligand/anti-ligand pairs as either a ligand or anti-ligand is arbitrary, when necessary to specify a particular component, a "ligand,” as used herein, is meant to describe peptides or proteins displayed on a genetic package carrying an expressible transgene. Further, when necessary to define anti-ligand with specificity, an "anti-ligand,” as used herein, demonstrates high affinity and is expressed on the surface of the target cell to be monitored for transgene expression.
  • a fragment of a ligand may be used within the present invention, so long as the fragment retains the ability to bind to the appropriate cell surface molecule.
  • ligands with substitutions or other alterations, but which retain binding ability may also be used.
  • a particular ligand refers to apolypeptide(s) having an amino acid sequence of the native ligand, as well as modified sequences (e.g., having amino acid substitutions, deletions, insertions or additions compared to the native protein), as long as the ligand retains the ability to bind to its receptor/cell surface moiety on an endothelial cell and be internalized.
  • Ligands also encompass muteins that possess the ability to bind to its receptor/cell surface moiety expressing cells and be internalized. Such muteins include, but are not limited to, those produced by replacing one or more of the cysteines with serine as described herein. Typically, such muteins will have conservative amino acid changes. DNA encoding such muteins will, unless modified by replacement of degenerate codons, hybridize under conditions of at least low stringency to native DNA sequence encoding the wild-type ligand.
  • DNA encoding a ligand may be prepared synthetically based on known amino acid or DNA sequence, isolated using methods known to those of skill in the art (e.g., PCR amplification), or obtained from commercial or other sources.
  • DNA encoding a ligand may differ from the above sequences by substitution of degenerate codons or by encoding different amino acids. Differences in amino acid sequences, such as those occurring among the homologous ligand of different species as well as among individual organisms or species, are tolerated as long as the ligand binds to its receptor.
  • Ligands may be isolated from natural sources or made synthetically, such as by recombinant means or chemical synthesis.
  • ligands may be either natural or synthetic ligands, including ligands possessing naturally-occurring polypeptide sequences and ligands with non- naturally-occurring polypeptide sequences.
  • ligands may be encoded by naturally-occurring nucleic acid sequences or non-naturally-occurring nucleic acid sequences.
  • a ligand used in the context of this invention retain any of its in vivo biological activities, other than its ability to bind a receptor/cell surface moiety on a cell and to be internalized.
  • a ligand may be desirable in certain contexts for a ligand to manifest certain of its biological activities, such as, for example, stimulation or inhibition of cell growth or proliferation, replication, or apoptosis, etc.
  • a ligand may display reduced or altered biological activities.
  • non- or reduced-mitogenic ligands can be constructed by swapping the receptor- binding domain with the receptor-binding domain of a related protein.
  • the domain of FGF2 may be swapped with the receptor-binding domain of FGF7 to create an FGF that does not cause proliferation and may alter the binding profile.
  • binding and intemalization may still be readily assayed by any one of the following tests or other equivalent tests.
  • these tests involve labeling the ligand, incubating it with target cells, and visualizing or measuring intracellular label.
  • the ligand may be fluorescently labeled with FITC or radiolabeled with 1 5 I, incubated with cells, and examined microscopically by fluorescence microscopy or confocal microscopy for intemalization. It will be apparent from the teachings provided within the subject application which of the biological activities are desirable to maintain.
  • Ligands include all binding partners for known and/or identified viral receptors/cell surface moieties, including those listed in Table I and described earlier, as well as any others.
  • ligands include peptides and polypeptides that bind cell surface moieties (e.g. receptors) upregulated directly or indirectly during any stage of microbial infection.
  • Suitable ligands also include all peptides and polypeptides capable of binding any cell surface moiety that displays a similar or overlapping pattern of expression with cells infectable by a viras.
  • Any known or identified receptor- or cell moiety-binding ligand may be used in the present invention. Any protein, peptide, analogue, peptidomimetic or fragment thereof that binds to a cell-surface moiety or receptor and is internalized may be used. Additionally, any such molecule that binds to a cell surface receptor that is upregulated in infected target cells may be used. Such a cell surface receptor should be expressed at least two-fold higher levels in infected cells, preferably at least five-fold higher, and most preferably at least ten-fold higher. In most preferred embodiments, an upregulated receptor/moiety is not detectably expressed in non-infected cells. Methods of determining the expression of cell surface receptors/moieties are well known in the art.
  • the ligands for attachment to delivery vehicles/packages may be produced by recombinant or other means known in the art.
  • the DNA sequences and methods to obtain the sequences of known ligands are well known, (see GenBank). Based on the DNA sequences, the genes may be synthesized either synthetically, amplified from cellular genomic DNA or cDNA, isolated from genomic or cDNA libraries, and the like. Restriction sites to facilitate cloning into the phage or phagemid vector may be incorporated, such as in primers used for amplification.
  • Antibodies to moieties present on the surface of cells are useful within the context of the present invention, particularly when the antibody is internalized following binding.
  • antibodies should be capable of specifically binding to a viral receptor or a receptor upregulated upon infection.
  • Such antibodies include, but are not limited to, antibodies to the receptors listed in Table I.
  • Antibodies that are specific to cell surface moieties expressed by cells are readily generated as monoclonals or polyclonal antisera. Many such antibodies are available (e.g., from American Type Culture Collection, Rockville, MD). Alternatively, antibodies to ligands that bind/internalize may also be used.
  • the phage particles will have antibody on their surface, which will then be complexed to the ligand (see further discussion below).
  • antibodies are understood to include monoclonal antibodies, polyclonal antibodies, anti-idiotypic antibodies, humanized antibodies, PrimatizedTM antibodies, and antigen binding antibody fragments (e.g. , Fab, and F(ab') 2 , F v variable regions, or complementarity determining regions), for example.
  • Antibodies are generally accepted as specific if they bind with a K ( j of greater than or equal to 10 _7 M, preferably greater than of equal to 10 _8 M.
  • amonoclonal antibody or binding partner can be readily determined by one of ordinary skill in the art (see Scatchard, Ann. N.Y. Acad. Sci. 51:660-672, 1949). Once suitable antibodies have been obtained, they may be isolated or purified by many techniques well known to those of ordinary skill in the art. Antibodies are produced by any means available in the art. For example, antibodies against cell surface molecules may be raised by immunization of mice, rats, rabbits or other animals with peptides, polypeptides, or cells (or membrane preparations thereof) expressing a cell surface receptor. Various immunization protocols may be found in for example, Harlow and Lane Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988 and Coligan etal.
  • Hybridomas are preferred (see, U.S. Patent Nos. RE 32,011, 4,902,614, 4,543,439, and 4,411,993; Harlow and Lane, supra; and Coligan et al , supra; for protocols).
  • Antibody-secreting hybridomas are grown, and the antibodies are tested for binding to the immunizing peptides, polypeptides, or cells by ELISA, section staining, flow cytometry, confocal microscopy and the like.
  • Positive antibodies are further tested for intemalization.
  • One assay that is used is a test for an antibody to kill cells. Briefly, the test hybridoma antibody and test cells are incubated. Unbound antibody is washed away. A second stage antibody, such as an anti-IgG antibody, conjugated to saporin is incubated with the test cells. Cell killing is assessed by any known assay, including trypan blue exclusion, MTT uptake, fluorescein diacetate staining, and the like.
  • portions or fragments, such as Fab and Fv fragments, of antibodies may also be constructed utilizing conventional enzymatic digestion or recombinant DNA techniques to yield isolated variable regions of an antibody.
  • the genes which encode the variable region from a hybridoma producing a monoclonal antibody of interest are amplified using nucleotide primers for the variable region.
  • techniques may be utilized to change a "murine" antibody to a "humanized” antibody, without altering the binding specificity of the antibody.
  • the invention includes the identification and selection of ligands and receptors/cell surface moieties capable of mediating infectious agent entry into a cell, including viral receptors and virus-associated receptors.
  • virus-associated receptors and “virus-associated moieties” include any viral receptor or moiety, any receptor or moiety upregulated during viral infection, and any receptor or moiety expressed predominantly in cells capable of being infected by a viras.
  • microbe-associated receptors/moieties include any microbe receptor/moiety, any receptor or moiety upregulated during microbe infection, and any receptor or moiety expressed predominantly in cells capable of being infected by a microbe.
  • phage are used to identify such ligands and moieties.
  • Phage have previously been used to display libraries of peptides or antibodies on their surface and to test for binding affinity to various targets. Such binding occurs primarily through electrostatic interactions between a displayed protein and the target, usually an immobilized protein, but cells and even organs have been used as targets. To date, however, there has been very little effort to exploit phage display technology as a way to identify cognate ligand/receptor and ligand/moiety pairs, particularly in mammalian systems.
  • receptors or other cell surface moieties involved in microbe binding and/or intemalization are identified using phage.
  • microbe-associated receptors/moieties are identified by screening a filamentous phage library that expresses on the phage surface polypeptides encoded by exogenous DNA sequences.
  • the exogenous DNA sequences are cDNAs generated from a cell infectable by a microbe of interest.
  • the exogenous DNA sequences are inserted in the gene III or gene VIII gene of the filamentous phage to produce a phage library.
  • Phage and phage libraries expressing infectable cell surface moieties may be screened by any method available in the art, including phage display and panning techniques, as described below. Phage expressing a cell surface moiety involved in binding to an infectious agent may also be selected by traditional and routine cell biology techniques such as affinity chromatography and binding assays to detect binding to a microbe or microbial ligand of interest. Full length cDNAs, genes, and polypeptides corresponding to receptor/moiety-related DNA fragments identified according to the invention may be isolated by routine molecular biology techniques, such as, for example, polymerase chain reaction and screening of cDNA libraries using the identified fragment as a probe.
  • Ligands may be identified and selected by any method available in the art, such as phage display, panning, bio-panning, or Ligand Identification Via Expression ("LIVETM").
  • Methods of phage display and panning techniques are widely known in the art and described in the literature (see, for example, U.S. Patent No. 5,223,409; and co- pending U.S. Patent Application Serial No. 60/057,067 "Methods using phage display for selecting internalizing ligands for gene delivery", filed 29 August 1997.) Briefly, in one method, DNA sequences are inserted into the gene III or gene VIII gene of a filamentous phage, such as Ml 3.
  • the inserted DNA sequences may be randomly generated, or they may be variants of a known binding domain for binding target cells. Single chain antibodies may readily be generated using this method. Generally, the inserts encode from 6 to 20 amino acids. The peptide encoded by the inserted sequence is displayed on the surface of the bacteriophage.
  • Bacteriophage expressing a binding domain for the target cells are selected for by binding to cells or intemalization of bacteriophage or, preferably, by expression of a detectable or selectable transgene encoded by the bacteriophage genome.
  • bacteriophage expressing a binding domain for the target cell may be selected for by binding to membrane preparations, peptides, or polypeptides corresponding to viral receptors or virally induced cell surface receptors.
  • the cells are grown in selective medium (e.g., containing drug) or isolated on the basis of expression (e.g., flow cytometry).
  • the insert DNA is recovered, generally by amplification of lysed cells, analyzed (e.g., DNA sequence analysis) and used in the present invention. Eluted phage are propagated in the bacteria host. In any of these methods, further rounds of selection may be performed to select for a few phage binding with high affinity. The DNA sequence of the insert in the binding phage is then determined. Once the predicted amino acid sequence of the binding peptide is known, sufficient peptide for use in chemical conjugate may be made by recombinant means or synthetically and by recombinant means for use as a fusion protein. The peptide may be generated as a tandem array of two or more peptides, in order to maximize affinity or binding.
  • LIVETM methods are described in detail in U.S. Patent Applications Serial No. 09/195,379, filed November 17, 1998, Serial No. 09/193,445, filed November 17, 1998, Serial No.09/258,689, filed February 26, 1999, and Serial No.09/866,073, filed May 24, 2001, entitled “Methods using genetic package display for selecting internalizing ligands for gene delivery", which are hereby incorporated in their entirety.
  • LIVETM permits the simultaneous identification of and selection for ligands capable of delivering a reporter or selectable marker to a cell, thereby providing for the identification of preferred ligands that bind and internalize.
  • Phage display methods are used to identify ligands or epitopes of infectious agents involved in attachment and/or intemalization into host cells.
  • an "epitope" is at least a portion of an infectious agent polypeptide that is capable of binding a receptor/moiety on a target cell.
  • a phage display library comprising cDNAs from an infectious agent is screened using cells capable of being infected by the infectious agent. These cells may be cells capable of being infected in vitro or in vivo, such as, for example, in an animal model of disease. In addition, the cells may be substantially pure (e.g. cell line) or they may mixed with other cell types, for example, in a tissue.
  • a phage display library comprising random peptides is screened using cells capable of being infected by an infectious agent.
  • Methods of producing phage display libraries comprising cDNAs and random peptides are well known in the art, and are described in the references cited above.
  • LIVETM methods are used to identify epitopes and ligands to cell surface receptors/moieties associated with infection, such as receptors and moieties up-regulated during viral or microbial infection, by infecting cells with an infectious agent, and subsequently screening for ligands that are internalized by the infected cells. Screening against the target cell or tissue can be performed in vitro or in vivo.
  • LINETM methods offer advantages over other phage display screening methods.
  • the criteria for a positive "hit” using LIVETM methods is that the phage must be able to bind, be internalized, and enable detection of the internalized ligand by detecting a selectable marker, such as, for example, by expressing a vector genomic D ⁇ A containing a reporter/selectable gene in the target cell or allowing direct nucleic acid detection (e.g. , PCR or D ⁇ A binding).
  • a selectable marker such as, for example, by expressing a vector genomic D ⁇ A containing a reporter/selectable gene in the target cell or allowing direct nucleic acid detection (e.g. , PCR or D ⁇ A binding).
  • the phage should bind, internalize, uncoat, translocate to the nucleus, and replicate, in order to express the gene or otherwise facilitate detection (however, translocation and uncoating may occur in any order).
  • only phage that reach the nucleus are selected.
  • LIVETM screens for epitopes and ligands are based on infectious function, rather than immunodominance, which does not necessarily produce a neutralizing immune response. By screening for epitopes or ligands that bind a portal of entry for an infectious agent, it is more likely that a ligand capable of directing entry of a delivery vehicle or an epitope capable of blocking infection is identified and selected.
  • An epitope or ligand may be identified by any available means, such as PCR, for example.
  • An initial selection of cells is performed using the detection of a reporter gene, selective conditions, or the like, and total D ⁇ A is recovered from these cells.
  • the recovered D ⁇ A is used as a template for PCR primers that are designed to flank the sequence of interest (the ligand encoding sequence, i.e., by using the pill or pVIII sequences flanking the ligand insert as primer templates).
  • the primers can be manufactured such that they can be easily removed from the reaction mixture, for example, the primers may contain a biotin moiety at the 5 '-end.
  • the primers may be removed by any known methods, including, for example, gel extraction, selective precipitation, and the like. In other alternatives primers need not be removed, however their removal facilitates ligation efficiency. Further, the primers can provide restriction sites for subcloning, etc. Following amplification, the PCR product is purified to remove the polymerase and digested with restriction enzyme to excise the putative ligand insert. Enzymes are chosen in order to facilitate directional subcloning into either the original vector or a new construct, if so desired. Following enzyme digestion, the extraneous sequences are removed (e.g. , by using biotinylated primers and streptavidin conjugated to beads or other solid support).
  • the resulting DNA sequences are then ligated into the desired vector and the resulting vector is transformed into bacteria using standard methodologies.
  • the nucleic acids encoding the identified epitope or ligand can be used to generate ligands for delivery vehicles and ligand displaying genetic packages described herein.
  • recovery of replicated internalized nucleic acid molecules may be achieved via a nucleic acid binding domain.
  • the phage genome can be altered such that a DNA binding sequence is incorporated therein.
  • the phage vector may contain one or more copies of the lac operon, thereby allowing any internalized and replicated phage vectors to be purified from a cell lysate by a solid surface having conjugated thereto the lac repressor protein.
  • target cells are contacted with ligand displaying genetic packages (e.g., phage) for 48 to 72 hours.
  • the replicated vector is double stranded and the double stranded form of the lac operon will bind the lac repressor.
  • the cells are then lysed and nuclear extracts are prepared, which are then passed over a solid support (e.g., Sepharose 4B) having conjugated thereto the lac repressor protein.
  • the column is washed, then eluted by a salt or pH gradient, thereby releasing the bound DNA which can now be utilized in PCR reactions to amplify the ligand sequences for sub-cloning into another vector for further rounds of infection or characterization or the DNA can be used directly (without PCR) to transform bacteria and thereby produce more phage for further screening.
  • Epitopes and ligands identified as described above may be used for a variety of purposes, including the production of ligand displaying genetic packages.
  • the identified epitopes and ligands are used to prevent infection by blocking binding or intemalization of an infectious agent.
  • an identified epitope or ligand is used as a vaccine to stimulate an immune response against the infectious agent.
  • the invention offers advantages for the production of vaccines. For example, since the immune response is generated against a ligand used by an infectious agent to gain entry into a cell, it is more likely that antibodies and antigen-presenting cells generated in response to the vaccine will prevent binding to and infection of a host cell.
  • immunodominant domains may mask or inhibit the generation of an immune response against other epitopes/moieties that may generate a more protective or more sustained immune response, such as epitopes/ligands involved in cell binding and/or intemalization.
  • the ligands for use herein may be customized for a particular application.
  • Means for modifying proteins is provided in the art. Briefly, additions, substitutions and deletions of amino acids may be produced by any commonly employed recombinant DNA method. Modified peptides, especially those lacking proliferative function, and chimeric peptides, which retain their specific binding and internalizing activities, are also contemplated for use herein. Modifications also include the addition or deletion of residues, such as the addition of a cysteine to facilitate conjugation and to form conjugates that contain a defined molar ratio (e.g., 1:1) of the polypeptides (see, e.g., U.S. Patent No. 5,175,147; PCT Application No.
  • Modification of the polypeptide may be effected by any means known to those of skill in this art.
  • the preferred methods herein rely on modification of DNA encoding the polypeptide and expression of the modified DNA.
  • DNA encoding one of the receptor-binding internalized ligands discussed above may be mutagenized using standard methodologies. For example, cysteine residues that are responsible for aggregate formation may be deleted or replaced. If necessary, the identity of cysteine residues that contribute to aggregate formation may be determined empirically, by deleting and/or replacing a cysteine residue and ascertaining whether the resulting protein aggregates in solutions containing physiologically acceptable buffers and salts. In addition, fragments of these cell surface moiety-binding internalized ligands may be constracted and used.
  • the binding region of many of these ligands have been delineated.
  • the receptor binding region of FGF2 has been identified by mutation analysis and FGF peptide agonists/antagonists to reside between residues 33-77 and between 102-129 of the 155 amino acid form (Baird et al., PNAS 85:2324; Erickson et al, Biochem. 55:3441).
  • Exons 1-4 of VEGF are required for receptor binding. Fragments may also be shown to bind and internalize by any one of the tests described herein.
  • Mutations may be made by any method known to those of skill in the art, including site-specific or site-directed mutagenesis of DNA encoding the protein and the use of DNA amplification methods using primers to introduce and amplify alterations in the DNA template, such as PCR splicing by overlap extension (SOE).
  • Site-directed mutagenesis is typically effected using a phage vector that has single- and double-stranded forms, such as Ml 3 phage vectors, which are well-known and commercially available.
  • Other suitable vectors that contain a single-stranded phage origin of replication may be used (see, e.g., Veira et al, Meth. Enzymol. 15:3, 1987).
  • site-directed mutagenesis is performed by preparing a single-stranded vector that encodes the protein of interest (i. e. , a member of the FGF family or a cytotoxic molecule, such as a saporin).
  • An oligonucleotide primer that contains the desired mutation within a region of homology to the DNA in the single-stranded vector is annealed to the vector followed by addition of a DNA polymerase, such as E. coli DNA polymerase I (Klenow fragment), which uses the double stranded region as a primer to produce a heteroduplex in which one strand encodes the altered sequence and the other the original sequence.
  • the heteroduplex is introduced into appropriate bacterial cells and clones that include the desired mutation are selected.
  • the resulting altered DNA molecules may be expressed recombinantly in appropriate host cells to produce the modified protein.
  • Suitable conservative substitutions of amino acids are well-known and may be made generally without altering the biological activity of the resulting molecule. For example, such substitutions are generally made by interchanging within the groups of polar residues, charged residues, hydrophobic residues, small residues, and the like. If necessary, such substitutions may be determined empirically merely by testing the resulting modified protein for the ability to bind to and internalize upon binding to the appropriate receptors. Those that retain this ability are suitable for use in the conjugates and methods herein.
  • an amino acid residue of a receptor-binding internalized ligand is non-essential if the polypeptide that has been modified by deletion or alteration of the residue possesses substantially the same ability to bind to its receptor and internalize a linked agent as the unmodified polypeptide.
  • Ligands or fragments thereof that bind to a cell-surface receptor/moiety and are internalized, but which are mimetics of "true" polypeptides, are also contemplated for use in the present invention.
  • the invention contemplates the preparation and use of non-peptide peptidomimetics useful for mimicking the activity of peptides, which makes peptidomimetics additional sources of targeting ligands that may be attached to the phage-based vectors of the present invention.
  • Peptidomimetics of antibodies are thus useful as disclosed herein, not only as ligands but as molecules useful in linking phage particles to targeting ligands.
  • Other useful peptidomimetic molecules useful as ligands and/or "linkers" herein are described in published PCT Publication No. WO 92/20704; Brandt, et al., Antimicrob Agents Chemother, 40:1078, 1996; Sepp-Lorenzino, et al, Cancer Res, 55:5302, 1995; and Chander et al, JPharm Sci, 84:404, 1995.
  • operative linkage or operative association of two nucleotide sequences refers to the functional relationship between such sequences.
  • Nucleotide sequences include, but are not limited to, DNA encoding a product, DNA encoding a signal sequence, promoters, enhancers, transcriptional and translational stop sites, and polyadenylation signals.
  • operative linkage of DNA encoding a ligand to a promoter refers to the physical and functional relationship between the DNA and the promoter such that transcription of the DNA is initiated from the promoter by an RNA polymerase that specifically recognizes, binds to, and transcribes the DNA.
  • Host organisms include those organisms in which recombinant production of heterologous proteins have been carried out, such as bacteria (for example, E. coli), yeast (for example, Saccharomyces cerevisiae and Pichiapastoris), mammalian cells, and insect cells.
  • bacteria for example, E. coli
  • yeast for example, Saccharomyces cerevisiae and Pichiapastoris
  • mammalian cells for example, a virus
  • insect cells for example, a virus, and insect cells.
  • preferred host organisms are E. coli bacterial strains.
  • the DNA construct encoding the desired protein is introduced into a plasmid for expression in an appropriate host.
  • the host is a bacterial host.
  • the sequence encoding the ligand is preferably codon-optimized for expression in the particular host.
  • a human ligand is expressed in bacteria, its codons would be optimized for bacterial usage.
  • the gene can be synthesized as a single oligonucleotide. For larger proteins, splicing of multiple oligonucleotides, mutagenesis, or other techniques known to those in the art may be used.
  • sequences of nucleotides in the plasmids that are regulatory regions, such as promoters and operators, are operationally associated with one another for transcription.
  • the sequence of nucleotides encoding the ligand may also include DNA encoding a secretion signal, whereby the resulting peptide is a precursor protein.
  • the resulting processed protein may be recovered from the periplasmic space or the fermentation medium.
  • the plasmids used herein include a promoter in operative association with the DNA encoding the protein or polypeptide of interest and are designed for expression of proteins in a bacterial host. Suitable promoters for expression of proteins and polypeptides herein are widely available and are well known in the art. Inducible promoters or constitutive promoters that are linked to regulatory regions are preferred. Such promoters include, but are not limited to, the T7 phage promoter and other T7-like phage promoters, such as the T3, T5 and SP6 promoters, the trp, lpp, and lac promoters, such as the lacUV5, from E.
  • coli the P10 or polyhedron gene promoter of baculovims/insect cell expression systems (see, e.g., U.S. Patent Nos. 5,243,041, 5,242,687, 5,266,317, 4,745,051, and 5,169,784) and inducible promoters from other eukaryotic expression systems.
  • promoters are inserted in a plasmid in operative linkage with a control region such as the lac operon.
  • Preferred promoter regions are those that are inducible and functional in E. coli.
  • suitable inducible promoters and promoter regions include, but are not limited to: the E. coli lac operator responsive to isopropyl ⁇ -D-thiogalactopyranoside (IPTG; ,yee, et al.Nakamuraet ⁇ /., Cell 75:1109-1117, 1979); the metallothionein promoter metal-regulatory-elements responsive to heavy-metal (e.g. , zinc) induction (see, e.g. , U.S. Patent No.
  • the plasmids also preferably include a selectable marker gene or genes that are functional in the host.
  • a selectable marker gene includes any gene that confers a phenotype on bacteria that allows transformed bacterial cells to be identified and selectively grown from among a vast majority of untransformed cells.
  • Suitable selectable marker genes for bacterial hosts include the ampicillin resistance gene (Amp r ), tetracycline resistance gene (Tc r ) and the kanamycin resistance gene (Kan 1 ). The kanamycin resistance gene is presently preferred.
  • the plasmids may also include DNA encoding a signal for secretion of the operably linked protein.
  • Secretion signals suitable for use are widely available and are well known in the art. Prokaryotic and eukaryotic secretion signals functional in E. coli may be employed. The presently preferred secretion signals include, but are not limited to, those encoded by the following E. coli genes: ompA, ompT, ompF, ompC, beta-lactamase, and alkaline phosphatase, and the like (von Heijne, J Mol. Biol. 184:99-105, 1985).
  • the bacterial pelB gene secretion signal (Lei et al, J.
  • Bacteriol 169:4379, 1987), the phoA secretion signal, and the cek2 functional in insect cell may be employed.
  • the most preferred secretion signal is the E. coli ompA secretion signal.
  • Other prokaryotic and eukaryotic secretion signals known to those of skill in the art may also be employed (see, e.g., von Heijne, J Mol. Biol. 184:99-105, 1985).
  • one of skill in the art can substitute secretion signals that are functional in yeast, insect or mammalian cells to secrete proteins from those cells.
  • the DNA plasmids also include a transcription terminator sequence.
  • the entire transcription terminator may be obtained from a protein- encoding gene, which may be the same or different from the inserted gene or the source of the promoter.
  • Particularly preferred plasmids for transformation of E. coli cells include the p ⁇ T expression vectors (see U.S PatentNo.4,952,496; available fromNovagen, Madison, WI).
  • Such plasmids include p ⁇ T 11a, which contains the T71ac promoter, T7 terminator, the inducible E. coli lac operator, and the lac repressor gene; p ⁇ T 12a-c, which contains the T7 promoter, T7 terminator, and the E.
  • coli ompT secretion signal and p ⁇ T 15b, which contains a His-TagTM leader sequence for use in purification with a His column and a thrombin cleavage site that permits cleavage following purification over the column, the T7-lac promoter region and the T7 terminator.
  • plasmids include the pKK plasmids, particularly pKK 223- 3, which contains the tac promoter (Pharmacia Biotech). Plasmid pKK has also been modified by replacement of the ampicillin resistance gene with a kanamycin resistance cassette (Pharmacia Biotech) .
  • Other plasmids include the pIN-IIIompA plasmids (see U. S . Patent No. 4,575,013), which have a cloning site linked in transcriptional reading frame with four functional fragments derived from the lipoprotein gene of E.
  • Baculovirus vectors such as pBlueBac (also called p JV ⁇ TL and derivatives thereof), particularly pBlueBac III (Invitrogen, San Diego, CA) may be used for expression of the polypeptides in insect cells.
  • a DNA construct may be made in a baculovirus vector and then co-transfected with wild type vims into sf9 insect cells from Spodoptera frugiperda (see, e.g., Luckow et al., Bio/technology t5:47-55, 1988, and U.S. Patent No. 4,745,051).
  • Expression vectors compatible with eukaryotic cells can also be used to form the recombinant nucleic acid molecules for use in the present invention.
  • Mammalian cell expression vectors are well known in the art and are available from several commercial sources. Typically, such vectors are provided containing convenient restriction sites for insertion of the desired DNA segment, and provide the signals required for gene expression in a mammalian cell.
  • Typical of such vectors are thepREP series vectors and EBVhis available from Invitrogen (San Diego, CA), the vectors pTDTl (ATCC #31255), pCPl (ATCC #37351) and pJ4W (ATCC #37720) available from the American Type Culture Collection (ATCC), and the like.
  • Successfully transformed cells i.e., cells that contain a nucleic acid molecule of the present invention, can be identified by well known techniques. For example, cells resulting from the introduction of an rDNA of the present invention can be subjected to assays for detecting the presence of specific rDNA using a nucleic acid hybridization method such as that described by Southern, J Mol.
  • the DNA fragment is replicated in bacterial cells, preferably in E. coli.
  • the preferred DNA fragment also includes a bacterial origin of replication.
  • Preferred bacterial origins of replication include, but are not limited to, the fl -ori and col El origins of replication.
  • Preferred hosts contain chromosomal copies of DNA encoding T7 RNA polymerase operably linked to an inducible promoter, such as the lacUV promoter (see U.S. Patent No. 4,952,496).
  • Such hosts include, but are not limited to, lysogens E. coli strains HMS174(DE3)pLysS, BL21(DE3)pLysS, HMS174(DE3) and BL21(DE3).
  • Strain BL21(DE3) is preferred.
  • the DNA fragments provided may also contain a gene coding for a repressor protein, which is capable of repressing the transcription of a promoter that contains a binding site for the repressor protein.
  • the promoter can be derepressed by altering the physiological conditions of the cell.
  • Preferred repressor proteins include, but are not limited to the E. coli lad repressor responsive to IPTG induction, the temperature sensitive ⁇ cI857 repressor, and the like.
  • the "plasmid” is phagemid pEGFP-Nl (Clontech; Palo Alto, CA), which contains a green fluorescent protein (GFP) gene under control of the CMV immediate-early promoter.
  • the CMV promoter is highly active in a large variety of mammalian cell lines; however, other mammalian cell promoters can be used. Examples of other useful promoters active in mammalian cells include viral promoters (e.g. retro viral LTRs, MMTV LTR, HIV LTR, SV40 early and late promoters, Bovine Papilloma Viras, BPV) or non- viral inducible promoters (e.g.
  • promoters include those that are constitutive (e.g. Beta Actin) or tissue-specific (e.g. alphafetoprotein (AFP), carcinoembryonic antigen (CEA), alpha actin, and Myo D). Additional useful promoters are described elsewhere herein.
  • the phagemid also includes an SV40 origin of replication to enhance gene expression.
  • SV40 origin of replication to enhance gene expression.
  • Other viral replication systems can also be used; for example, EBV origin and EBNA or BPV are useful as disclosed herein.
  • a preferred E. coli strain in which to propagate phagemids is the E. coli host strain DH5 ⁇ F'.
  • Useful systems according to the present invention may further benefit from the use of helper bacteriophage.
  • the Ml 3 helper Ml 3K07 is particularly useful in conjunction with the aforementioned bacterial strains.
  • Methods of preparing phagemid particles are known in the art and may appropriately be modified depending on the system utilized, as those of skill in the art will appreciate. (See, e.g. , Sambrook et al. , Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, 1989; Rider et al, in Phage Display of Peptides and Proteins: A Laboratory Manual, Academic Press, 1996).
  • phage are used herein as a protypical example, nevertheless, it should be understood that methods described herein are applicable to the construction of any delivery package.
  • phage for example, present a ligand that is part of a fusion protein, direct chemical linkage, by sandwich, or charge- charge interactions. Exemplary methods for modifying bacteriophage to present ligands are described below.
  • Ligands are prepared as discussed herein by any suitable method, including recombinant DNA technology, isolation from a suitable source, purchase from a commercial source, or chemical synthesis.
  • Various preferred methods and modifications to ligands that may facilitate the linkage between the ligand and the phage protein are disclosed in published PCT Publication No. WO 96/36362.
  • Ligands may be associated with any filamentous phage protein or nucleic acid, provided the associated ligand is capable of binding to a cell surface receptor.
  • preferred filamentous phage proteins for ligand linkage include coat proteins, such as those encoded by genes VIII, III, VI, VII, and IX.
  • DNA encoding the polypeptide ligands may be isolated, synthesized or obtained from commercial sources or prepared as described herein. Expression of recombinant polypeptides may be performed as described herein; and DNA encoding these polypeptides may be used as the starting materials for the methods herein. DNA may be prepared synthetically based on the amino acid or DNA sequence or may be isolated using methods known to those of skill in the art, such as PCR, probe hybridization of libraries, and the like or obtained from commercial or other sources.
  • Fusion proteins of the present invention preferably comprise a gene encoding all or a receptor-binding polypeptide portion of a ligand genetically fused or linked to the coat protein-encoding gene of a bacteriophage particle using methods known to those of skill in the art (see, e.g., Smith and Scott Meth Enymol, 217:228-257, 1993; Kay, Phage Display of Peptides and Proteins: A Laboratory Manual, Academic Press, 1996).
  • “genetically fused” indicates that regions of two genes, or portions thereof, are physically combined, via recombinant DNA techniques, so that the resulting polynucleotide sequence encodes a fusion polypeptide comprising regions encoded by each gene.
  • the phage is a filamentous phage; Ml 3 is described herein as an exemplary preferred embodiment. Preparation of a fusion protein comprising Ml 3 gene III or VIII coat protein is described herein.
  • ligand-phage coat fusion proteins are the predominant species.
  • the copy number of ligand-phage coat fusions relative to wild type coat protein may be readily controlled by displaying the fusion at high copy number (type 3 or type 8 vectors) or at low copy number (type 3+3 or type 8+8 vectors).
  • Nucleotide sequences for the ligands are readily available (e.g., from GenBank) or may be synthesized or isolated by standard techniques. The ligand coding regions are inserted into the phage vectors using well-known methods.
  • Nucleotide sequences encoding ligand-phage fusions may also be further modified via the insertion of a mammalian reporter gene, in order to further verify binding and intemalization, as well as expression of the nucleic acid payload.
  • a mammalian reporter gene is EGFP; others include the sequences for ⁇ -galactosidase, luciferase, human growth hormone (HGH), and secreted alkaline phosphatase. Methods of preparing and using such sequences as described herein are known to those of skill in the art. (See, e.g., Sambrook et al, Molecular Cloning : A Laboratory Manual, Cold Spring Harbor Press, 1989.)
  • the expression cassette also includes an origin of replication.
  • the SN40 ori is particularly useful for generating high copy number replication in cell lines containing T antigen (e.g. , COS cells). A variety of useful methods and protocols are available which employ this and other origins of replication (see, e.g., Sambrook et al, Id).
  • Ligand-coat protein fusions may be tested for phage binding to the relevant receptor (e.g. a cognate receptor to the ligand) via known methods such as ELISA. Binding and intemalization of the fusion is readily assayed via art-recognized methods such as immunohistochemistry (Barry et al, Nature Med. 2: 299-305, 1996; Li, Nature Biotech. 15: 559-563, 1997).
  • the modified phage may further be tested for transduction of the reporter gene into the cells, preferably mammalian cells, by adding the phage to the cell cultures for a predetermined amount of time; depending on the protocol and reporter used, the number of cells expressing the reporter gene product at a subsequent, predetermined time is readily assessed using known methods.
  • the reporter gene is EGFP, which is translated into green fluorescent protein (GFP) when gene delivery and expression occur, one may readily measure the number of autofluorescing cells several hours later. Results are readily quantitated.
  • fluorescent reporters are used, flow cytometry is particularly useful to measure the distribution of cells expressing the fluorescent reporter gene in the population and by fluorometry of cell extracts to measure total amounts of reporter protein expressed.
  • a variation of the aforementioned procedure involves the construction of a fusion protein including all or a portion of an antibody molecule and a phage protein, thereby producing a phage protein-antibody conjugate.
  • preparation of a single- chain antibody-phage coat protein fusion is described as exemplary, other antibodies and fragments thereof are also useful as disclosed.
  • an antibody or portion thereof e.g., a mAb, Fab, or ScFv
  • a mAb, Fab, or ScFv is either prepared via direct synthesis (e.g. , using splice site overlap extension PCR, with the optional addition of restriction sites at the 5' and 3' ends of the sequence), or it is selected and isolated from ascites and is thereafter purified via known methods, such as affinity chromatography. If only the Fab fragment is to be used, an antibody harvested from the ascites is further modified according to standard protocols to produce the Fab, e.g. , through the use of papain digestion.
  • One exemplary antibody that specifically targets the FGF receptor is the 11 A8 antibody derived from the 11 A8 hybridoma.
  • 11 A8 recognizes ECDR1 by Western blot, can immunoprecipitate FGFRs from extracts of SK-HEP-1 and SK-MEL-28, a melanoma cell.
  • Antibody 11 A8 also stains SK-HEP-1 cells on the cell surface.
  • Receptor- binding fragments and variants of 11A8 and similar, receptor-binding antibodies, preferably, antibodies that also internalize may also be non-covalently (or covalently) attached to the phage coat in order that they may function as useful ligands, as described herein.
  • Other antibodies (and fragments thereof) that specifically target cellular receptors preferably those antibodies and fragments thereof that bind and internalize may be identified and synthesized according to the methods disclosed herein and via use of other methods described in the art.
  • Amplified heavy and light chain product is cloned into the expression vector.
  • the expression vector chosen preferably contains appropriate promoters and convenient restriction sites, as well.
  • the resultant protein may optionally be expressed as both N-terminal and C-terminal fusion protein.
  • flexible linkers may be added between the antibody and the ligand to help favor proper protein folding. Use of smaller antibody fragments, e.g. Fab or ScFv, likely facilitates folding even further.
  • the expression, purification and evaluation of antibody-phage fusion proteins is readily accomplished in host cells, e.g., in bacteria, using known protocols. Alternatively, one may fuse two monospecific antibodies or immunologically active portions thereof with different phage coat proteins, wherein each antibody is directed against the same receptor, or against different receptors.
  • Such a construct may be useful in situations in which the expressed cellular receptors are polymorphic and the use of phage vectors targeted to two or more of said polymorphisms would have a higher likelihood of delivering payload to the desired cells.
  • Preparation of a fusion between phage coat protein and bispecific antibodies is also within the scope of the present invention.
  • delivery vehicles targeted to one or more receptors may readily be prepared according to the within-disclosed methods.
  • the fused antibody portion(s) are directed to other ligands as opposed to specific receptors, rather than targeting the antibody/ies to the relevant receptors directly.
  • Such a procedure may be particularly useful in situations in which a ligand' s ability to bind its cognate receptor is dependent upon the ability of the ligand to attain a specific secondary or tertiary structure that would not be readily achievable if the ligand were incorporated into a ligand-phage fusion protein.
  • chimeric antibodies including receptor-binding ligands in place of their constant region are useful as disclosed herein.
  • bispecific antibodies generated as described in published UK Patent App. No. GB 2197323 are also preferred for use as described herein.
  • bridging antibodies such as those described in published PCT Publication No. WO 92/08801 may be useful in the treatment of conditions in which a "timed-release" of therapeutic nucleotide sequences is desired.
  • bispecific antibodies which link the vector to the target cell pending the co- or subsequent administration of vectors carrying highly specific proteases, ribozymes or deoxyribozymes that release the bound vectors, thereby freeing them to bind directly to their targeted receptor and internalize the vector, is also contemplated within the scope of the present invention.
  • the recombinant fusion protein must bind the cognate receptor. Fusion proteins may be analyzed for their binding capacity in an ELISA, according to known techniques. To test the functionality of the receptor binding domain, binding and intemalization assays may be performed on receptor-positive cells and binding specificity is determined by including unlabeled fusion protein as a competitor per standard protocols.
  • intemalization of a fusion protein is determined by preincubating receptor-positive cells with labeled fusion protein, washing cells to remove unbound labeled protein, and conducting further incubations at predetermined temperatures and for various time intervals to allow receptor intemalization. Following the removal of surface-bound radiolabeled protein, cells are lysed and radioactivity is determined in the cell lysate. The analysis will determine the capacity of the fusion protein to bind its cognate receptor in the context of a fusion protein. In order to facilitate the attachment of a ligand sequence to a phage sequence, the phage protein may be modified at the molecular level.
  • a nucleotide sequence encoding a therapeutic molecule, toxin, or other regulatory molecule may be operatively linked for expression to a phage nucleotide sequence, particularly to a sequence encoding a structural protein.
  • protein III or protein NIH of filamentous phage e. g. M 13
  • one or more heterologous sequences may be inserted at an internal site, i. e. , within the phage coat protein sequence.
  • Various methods of preparing such fusions are available in the art and are contemplated by the present invention.
  • phage proteins may be modified via means that are not precisely "immunologic" or “genetic.” Modification of phage proteins via means other than those exemplified herein is fully within the scope of the present invention.
  • useful filamentous phage-based vectors of the present invention may undergo chemical alteration of their coat proteins, e.g., in a manner that affects the vector's immunogenicity, in order to regulate the uptake and persistence of the vector in the cells of an individual to whom the within-disclosed therapeutic constructs and compositions are administered.
  • Methods of making such alterations to proteins via chemical and physical means are known to those of skill in the art and may readily be ascertained in the relevant literature.
  • bispecific antibodies or fragments thereof e.g. , in the form of bispecific ScFv's
  • various techniques summarized above e.g. with respect to the preparation of an ScFv
  • a fusion protein comprising phage coat protein and a monospecific antibody is useful in linking a phage vector to a ligand.
  • an antibody or fragment thereof is readily understood in the art; also see Section B above.
  • the antibody portion of the antibody-fusion protein is preferably raised against a preselected ligand, e.g., one that is not easily incorporated into a fusion protein, perhaps due to corrfbrmational difficulties.
  • a preselected ligand e.g., one that is not easily incorporated into a fusion protein, perhaps due to corrfbrmational difficulties.
  • Such an antibody-phage coat fusion may then be utilized to bind a ligand that targets a specific receptor to the phage vector, for the purpose of delivering a payload to a cell expressing the relevant cognate receptor.
  • One method for targeting the phage to cellular receptors is to link the phage to the ligand via avidin-biotin, which may be performed essentially as follows.
  • a ligand- phage complex is assembled in the presence of test cells at 0°C, and followed by incubation at 37°C to allow intemalization.
  • a ligand molecule is conjugated to biotin at the single free sulfhydryl group using biotin-BMCC (Pierce; Rockford, IL) according to the manufacturer's suggested protocol. Unreacted biotin is removed by passing the reaction over a PD- 10 desalting column.
  • Cultured cells e.g., COS cells
  • a predetermined time period e.g. 24 hours
  • Cells are washed and biotinylated ligand is added (often on ice) thereafter, and the preparation is incubated for a predetermined period of time.
  • Avidin e.g. Neutravidin, a neutral carbohydrate-stripped avidin; Pierce, Rockford, IL
  • Biotinylated anti-phage antibody such as anti-M13 antibody when the phage is M13, is then added and incubated for a predetermined time period.
  • the cell wash is then repeated and a sufficient quantity of colony forming units are added to each receptacle or well, followed by further incubation, preferably on ice.
  • the cells are washed again, resuspended in fresh media and are allowed to incubate further for an appropriate period of time and at a predetermined temperature.
  • Expression of the agent encoded by the therapeutic nucleic acid sequence transported by the phage vector is thereafter monitored.
  • the vector further comprises a reporter gene, such as EGFP, which may readily be monitored via fluorescence microscopy or via a fluorescence activated cell sorter (FACS), according to standard techniques.
  • FACS fluorescence activated cell sorter
  • a ligand may alternatively be linked to a polycation such as polylysine, which then binds to phagemid particles as a result of charge-charge interactions between the positively-charged polycation and negatively-charged phage.
  • a ligand is covalently linked to polylysine using S-2-pyridyl disulfide (SPDP) according to known protocols (see, e.g. , Sosnowski etal. J. Biol Chem. 271: 33647-33653, 1996).
  • SPDP S-2-pyridyl disulfide
  • Phagemid particles are then mixed with ligand-polylysine or polylysine alone and allowed to stand at room temperature for a predetermined period of time prior to testing the conjugates in cultured cells, or prior to ex vivo or in vivo administration.
  • a reporter sequence may be included in the ligand-polylysine-phage construct; fluorescent markers such as GFP and fluorescein are particularly useful for in vitro assays.
  • the treated cells are examined by conventional means, e.g. fluorescent microscopy or FACS.
  • reporter sequence is EGFP
  • autofluorescent GFP positive cells are then counted in order to confirm that the GFP gene (and nucleotide sequences appended thereto) has been successfully transduced into the cells via phage linked to the ligand via polylysine. This method of phage transduction is an attractive alternative to the use of the avidin-biotin system described herein.
  • a bacteriophage preferably a filamentous bacteriophage
  • the coat proteins of a bacteriophage may be conjugated directly to a ligand using heterobifunctional crosslinking reagents.
  • a free lysine at the N-terminus of the gene VIII coat protein of Ml 3 phage is available for chemical modification (Armstrong et al, EMBO J. 2: 1641-1646, 1983) and may conveniently be employed for that purpose.
  • the procedure may be described as follows.
  • Phage particles are first thiolated, e.g., via the addition of SPDP, for a predetermined period of time and at a predetermined temperature. Unreacted reagent is removed; the ligand is then reacted with thiolated phage. Free ligand is removed, and the phage linked to the ligand are further purified according to standard protocols.
  • SATA N-succinimidyl S-acetylthioacetate
  • suitable heterobifunctional chemical reagents may be used to introduce the thiol function, rather than SPDP, pursuant to art-recognized methods.
  • the selected linker or linkers is (are) linked to the receptor-binding internalized ligands by chemical reaction, generally relying on an available thiol or amine group on the receptor-binding internalized ligands.
  • Heterobifunctional linkers are particularly suited for chemical conjugation and include such molecules as m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester, N-succinimidyl-(4- iodoacetyl)amino-benzoate, andN-succimmidyl-3-(2-pyridyldithio)propionate. (See, e.g., published UK Pat. App. Nos. GB 2268492, 2253626, and 2257431, the disclosures of which are incorporated by reference herein.)
  • the linkage of a ligand to phage protein is readily confirmed by polyacrylamide gel electrophoresis and immunoblotting of phage proteins.
  • the ligand-modified gene VIII protein is shifted from its apparent molecular mass in unlinked form to a significantly greater apparent molecular mass when linkage has been successful.
  • the addition of reducing agent to the sample buffer disrupts the disulfide linkage and yields free ligand and gene VIII protein.
  • the chemically modified ligand-phage constructs may then be assayed for their ability to transduce mammalian cells.
  • Ligand-modified phagemid particles bearing an expression cassette containing a promoter and a reporter sequence are added to plated cells in multiwell plates, for example.
  • the cells are assayed for marker protein expression at a predetermined interval following the addition of the ligand-phage constructs.
  • Another means of chemically linking a ligand to a phage particle comprises the expression of a specific reactive moiety on the surface of a phage particle, wherein that moiety is then conjugated specifically and directly to the selected ligand.
  • reactive moieties that may readily be engineered into the surface protein of a phage particle include various binding proteins, protein A, cysteine, and a wide variety of reactive groups, to name but a few examples. (See, e.g., published UK Pat. App. Nos. GB 2268492 and 2257431 , the disclosures of which are incorporated herein by reference.)
  • Still other ligands can be attached to the phage surface coat proteins.
  • the size of peptides/polypeptides attached by any means to phage coat proteins is highly variable, from small random peptides to full length proteins. Small random peptides of from 6 to 38 amino acids in length that bind cell surface receptors have been described (Scott and Smith, Science, 249: 386-390, 1990; Kay, Gene 128: 59-65, 1993).
  • protein molecules such as antibody Fab fragments ( ⁇ 5 OkDa) have been fused to phage coat proteins.
  • Modified Phage are propagated by any methods known in the art, including the method described by Sambrook et al. Suitable host bacteria that carry the F' episome are grown from an isolated colony to mid-log phase growth. Bacteriophage isolates can be picked from plaques that form on a lawn of infected host cells grown on semi-solid medium. The turbid plaques are slower growing infected cells that are visible to the naked eye against a lawn of more dense uninfected bacteria. Phage stocks can be prepared in liquid culture from well isolated plaques. About 1/10 of the phage from a single plaque are used to infect 50 ⁇ l of host bacteria in 2 ml of medium. The culture is incubated at 37°C
  • the bacteria are pelleted by microcentrifugation and the supernatant transferred to a fresh tube.
  • the titer of the phage in the supernatant should be about 10 12 pfu/ml and can be stored at 4°C or indefinitely at -20°C.
  • the present invention comprises delivering genetic payloads specifically to cells infected with an infectious agent/microbe, such as a virus, as well as cells at risk of or prone to infection.
  • the genetic payload is an anti-microbial agent, such as an anti- viral agent.
  • An anti-microbial agent is any molecule that inhibits or decreases replication, infectivity, or microbial count by an amount or degree sufficient to attenuate at least one pathological affect of infection by at least a detectable degree, preferably by at least 10% or preferably by at least 25%.
  • an anti-viral agent is any molecule or composition that inhibits or decreases viral replication, viral infectivity, rate of infection, and/or viral count by an amount or degree sufficient to attenuate at least one pathological affect or symptom of infection by at least a detectable degree, preferably by at least 10% or preferably at least 25%.
  • Pathological affects include any symptom or pathologic symptom associated with infection, including both short term and long term symptoms or complications, for example.
  • a genetic payload comprises a therapeutic nucleic acid molecule or gene.
  • genetic payloads are selected, in part, based upon their ability to enhance immune responsiveness or immobilization of the immune system.
  • payloads are selected, in part, based upon their ability to block or slow the rate of disease progression or viral replication.
  • the best predictor of disease outcome is immune status during progression. Patients with delayed responsiveness more often have fatal outcomes. Therefore, a genetic payload that slows the progression of infection allows time for immune system mobilization and maturation, significantly reducing mortality. This provides a proactive, post-infection intervention.
  • Therapeutic vaccines would likely either take too long to be effective or would not be effective at all in a patient already highly stimulated immunologically by the pathogen. However, such a platform could also complement a prophylactic vaccine program where protection decreased with time.
  • genetic payloads There are other potential benefits of using genetic payloads.
  • the products of genetic payloads i.e. the therapeutic protein or nucleic acid
  • Genetic payloads can remain elevated throughout treatment, unlike small molecule drags that cycle through peaks and troughs of concentration as each dose is given.
  • Genetic payloads can remain present for up to several weeks, so fewer treatments are necessary.
  • the treatments are concentrated at the site of pathology (i.e. the infected cell). And, these treatments are less likely to be circumvented by viral mutation.
  • a "therapeutic nucleic acid” or “therapeutic gene” describes any nucleic acid molecule used in the context of the invention that effects a treatment, or confers a reduction in either the number of infectious agents or a clinical symptom associated with infection to a target cell, animal, or plant. It includes, but is not limited to, nucleic acids encoding a peptide, a protein, a ribozyme, an antisense nucleic acid, a DNA intended to form triplex molecules, protein binding nucleic acids, and small nucleotide molecules.
  • the product of the therapeutic gene may be peptides, polypeptides, DNA or RNA. Examples of polypeptide therapeutic gene products include antibodies, intrabodies, and cytokines.
  • Therapeutic nucleic acids may be naturally-derived sequences or recombinantly derived.
  • a therapeutic nucleic acid may act directly or it may act indirectly by delivering a product with therapeutic properties.
  • Such a product may be a nucleotide or polypeptide, such as an antisense RNA or a cytokine, for example.
  • the therapeutic nucleic acid may encode all or a portion of a gene, and may function by recombining with DNA already present in a cell. It may also encode a portion of a protein and exert its effect by virtue of co-suppression or repression of a gene product. Similarly, it may encode a mutated protein, or fragment thereof, and function as a dominant-negative repressor of a corresponding wild-type protein.
  • the genetic payloads and therapeutic nucleotide compositions of the present invention are from about 12 base pairs to about 100,000 base pairs in length.
  • the nucleic acid molecule is from about 50 base pairs to about 50,000 base pairs in length. More preferably the nucleic acid molecule is from about 50 base pairs to about 10,000 base pairs in length. Even more preferably, it is a nucleic acid molecule from about 50 pairs to about 4,000 base pairs in length.
  • the therapeutic nucleic acid is provided in operative linkage with a selected promoter, and optionally in operative linkage with other elements that participate in transcription, translation, localization, stability and the like.
  • Such nucleic acid compositions may be delivered by a delivery vehicle, such as a bacteriophage, of this invention by a variety of methods including, for example, in vitro, in vivo, and ex vivo transduction methodologies.
  • Therapeutic nucleic acids of the invention that function by slowing viral progression may target any stage of viral progression and any viral or cellular polypeptide that functions in viral progression.
  • Stages of viral replication have been loosely categorized as initiation, replication, and release. Initiation is the stage in which the genetic material of the vimses is introduced into the cell and includes the steps of attachment, penetration, and uncoating.
  • Replication includes the steps of genome synthesis, RNA production, and protein synthesis and involves numerous viral proteins, such as those that ensure replication of the genome, package the genome into vims particles, and alter the metabolism of the infected cell so that vimses are produced. In a few instances, such as parvoviras, cellular enzymes replicate the viral genome, but in most cases, viral proteins are primarily responsible for genome replication.
  • the final stage of viral progression is release of viral particles from the infected cell. This stage includes the steps of assembly, maturation, and exiting, all of which are primarily controlled by viral proteins. Disrupting replication is a means of halting viral progression, thereby allowing a host's immune system more opportunity to ward off the infection. Interfering with viral initiation and release of particles are potential means of blocking infection of additional cells.
  • Specific targets for therapeutic nucleic acids include both coding regions and regulatory regions of viral genomes. In addition, cellular genes associated with viral replication may also be targets.
  • suitable targets include, but are not limited to, viral genes involved in transcription or translation, such as reverse transcriptase and RNA polymerase, gag, pol, env, rev, tat, and viral genes involved in host/pathogen interactions, such as p28 from ectromelia virus (ECTV).
  • viral genes involved in transcription or translation such as reverse transcriptase and RNA polymerase, gag, pol, env, rev, tat, and viral genes involved in host/pathogen interactions, such as p28 from ectromelia virus (ECTV).
  • ECTV ectromelia virus
  • Viral genes that encode cytokines, transcription regulators, nucleic acid binding proteins, immunomodulators, coat proteins, capsid proteins, envelope proteins, and metabolic regulators are all suitable targets, according to the invention.
  • HCV hepatitis C viras
  • HCV genome is approximately 9.6 kb in length
  • proteome encoded is a polyprotein of a little more than 3000 amino acid residues. This polyprotein is processed by a combination of host and viral proteases into structural and non-stractural proteins. Any of these proteins involved in replication, and homologs thereof, are suitable targets.
  • Polynucleotides encoding or regulating expression of key viral enzymes including the NS3 protease, NS3 helicase, and NS5b RNA-dependent RNA polymerase, are examples of targets, as are the enzymes themselves.
  • Targets also include a variety of cellular genes involved in cellular processes, particularly those related to cell proliferation, DNA replication, cell cycle, immune response, and apoptosis, including, for example, cytokines, transcription regulators and transcription factors, nucleic acid binding proteins, immunomodulators, immune response proteins, metabolic regulators, kinases, cyclins, and caspases.
  • Both viral and host proteins involved in gene expression, as well as viral genome regulatory elements are preferred targets for disruption.
  • studies of coronoviras replication have demonstrated that the viruses 3 ' untranslated region (UTR) plays a role in viral RNA synthesis, and both the viral poly(A) tail and host proteins such as poly(A)-binding protein (PABP) that specifically bind the viral 3' UTR have been implicated in viral RNA synthesis.
  • UTR untranslated region
  • PABP poly(A)-binding protein
  • the Ebola vims genome consists of a single negative strand of RNA that is non-polyadenylated, with a linear arrangement of genes, with some occurrence of overlap.
  • the order of the genes is: 3'-untranslated region, nucleoprotein, viral structural protein, VP35, VP40 glycoprotein, VP30, VP24, RNA dependent RNA polymerase (L), and 5'- untranslated region.
  • the viras transcribes its RNA and replicates in the cytoplasm of the infected cell. Replication is mediated by the synthesis of an antisense positive RNA strand that will serve as a template for additional viral genomes.
  • the Ebola (+) leader R ⁇ A sequence contains a potential stem-loop stmcture and has been postulated to play a role in gene expression (perhaps by altering ribosome binding). For example, the hairpin shape of the (+) leader strand may be conducive to nucleoprotein binding, and the subsequent conformational change may either promote replication or transcription.
  • Polynucleotide sequences encoding or regulating expression of the Ebola genes and polypeptides are all potential targets, according to the present invention. Genomic overlap regions may represent particularly desirable targets, as multiple genes may be disrupted simultaneously. In addition, nucleic acids capable of disrupting the potential Ebola leader R ⁇ A sequence stem-loop structure may prove a desirable target, as they inhibit nucleoprotein binding and/or replication or transcription.
  • the smallpox virus genome contains more than 200 genes. Genome replication is believed to involve self-priming, leading to the formation of high molecular weight concatemers (isolated from infected cells) that are subsequently cleaved and repaired to make viras genomes.
  • Gene expression is carried out by viral enzymes associated with the core and is divided into two phases: (1) the early genes, which are expressed before genome replication; and (2) the late genes, which are expressed after genome replication, since the late gene promoters are dependent on D ⁇ A replication for activity.
  • Early genes involved in replication represent preferred targets for disruption according to the invention, since disruption of such genes inhibits both genome replication and expression of the late genes.
  • a virus-encoded enzyme involved in replication is thymidine kinase.
  • the invention includes genetic payloads specific for an individual virus or infective agent
  • the genetic payload provides a molecule effective against two or more viruses or infective agents.
  • the genetic payload is effective against an entire class, family, or genus of viras or microbe. More preferably, the delivered genetic payload is effective against multiple classes of viruses or microbes.
  • the invention contemplates genetic payloads that target common cellular or conserved microbial nucleic acids or gene products.
  • Such genetic payloads are advantageous, because they allow the production of numerous viras or agent specific compositions, based upon a single genetic payload platform. Thus, additional composition can be produced rapidly, merely by introducing a ligand specific for the targeted vims or agent. 7.
  • Antisense and ribozymes are advantageous, because they allow the production of numerous viras or agent specific compositions, based upon a single genetic payload platform. Thus, additional composition can be produced rapidly, merely by introducing a ligand specific for the targeted vim
  • the vectors and vehicles provided herein may be used to deliver a ribozyme, antisense, and the like to targeted cells.
  • Such products include antisense RNA, antisense DNA, ribozymes, deoxyribozymes, triplex-forming oligonucleotides, and oligonucleotides that bind proteins.
  • the delivered nucleic acid molecule directs degradation of a targeted nucleic acid molecule.
  • Delivered nucleic acids can also include RNA trafficking signals, such as viral packaging sequences (see e.g., Sullenger et al. Science 262:1566, 1994).
  • Antisense nucleotides are oligonucleotides that bind in a sequence-specific manner to nucleic acids, such as mRNA or DNA. Antisense oligonucleotides may be of various length, including e.g. 12-25 nucleotides and up to several hundred nucleotides. Antisense oligonucleotides between 12 and 50 nucleotides long are frequently used. When bound to mRNA that has complementary sequences, antisense prevents translation of the mRNA (see, e.g., U.S. PatentNos. 5,168,053; 5,190,931; 5,135,917; 5,087,617).
  • Triplex molecules refer to single DNA strands that bind duplex DNA forming a co linear triplex molecule, thereby preventing transcription (see, e.g., U.S. Patent No. 5,176,996).
  • Particularly useful antisense nucleotides and triplex molecules are molecules that are complementary or bind to the sense strand of DNA or mRNA that encodes a protein involved in viral replication, such as those described above.
  • a ribozyme is an RNA molecule that specifically cleaves RNA substrates, such as mRNA, resulting in inhibition or interference with cell growth or expression. There are at least five known classes of ribozymes involved in the cleavage and/or ligation of RNA chains.
  • Ribozymes can be targeted to any RNA transcript and can catalytically cleave that transcript (see, e.g., U.S. PatentNo. 5,272,262; U.S. PatentNo. 5,144,019; and U.S. Patent Nos. 5,168,053, 5,180,818, 5,116,742 and 5,093,246).
  • ribozyme used to treat viral infection is the "hairpin" ribozyme gene engineered by Flossie Wong-Staal and colleagues at the University of California at San Diego.
  • An infected CD4 cell expressing this ribozyme generates short, hairpin-shaped RNA sequences ("hairpin" ribozymes) that selectively attack viral RNA, cleaving the strand.
  • hairpin ribozymes short, hairpin-shaped RNA sequences
  • Nucleic acids and oligonucleotides for use as described herein can be synthesized by any method known to those of skill in this art (see, e.g. , WO 93/01286, U.S. Application Serial No. 07/723,454; U.S. Patent Nos. 5,218,088; 5,175,269; 5,109,124).
  • Identification of oligonucleotides and ribozymes for use as antisense agents and DNA encoding genes for targeted delivery for genetic therapy involve methods well known in the art. For example, the desirable properties, lengths and other characteristics of such oligonucleotides are well known.
  • Antisense oligonucleotides may be designed to resist degradation by endogenous nucleolytic enzymes using linkages such as phosphorothioate, methylphosphonate, sulfone, sulfate, ketyl, phosphorodithioate, phosphorarnidate, phosphate esters, and the like (see, e.g., Stein in: Oligodeoxynucleotides. Antisense Inhibitors of Gene Expression, Cohen, Ed, Macmillan Press, London, pp. 97-117, 1989); Jager et al, Biochemistry 27:7237, 1988).
  • linkages such as phosphorothioate, methylphosphonate, sulfone, sulfate, ketyl, phosphorodithioate, phosphorarnidate, phosphate esters, and the like (see, e.g., Stein in: Oligodeoxynucleotides. Antisense Inhibitors of Gene Expression, Cohen, Ed, Mac
  • Expression vectors useful in such embodiments are also contemplated herein. (See, e.g., Published International Application Nos. WO 92/06693, WO 96/17086, and WO 95/31551; and U.S. Patent Nos. 4,987,071, 5,580,967 and 5,595,873, the disclosures of which are incorporated by reference herein). Methods of engineering such expression vectors are well known and available in the art.
  • a method of forming an enzymatic RNA molecule expression vector includes providing a vector comprising nucleic acid encoding a first ribozyme and providing a single-stranded DNA molecule encoding a second ribozyme.
  • the single-stranded DNA is then allowed to anneal to form a partial duplex DNA, which can be filled in by treatment with an appropriate enzyme, such as a DNA polymerase in the presence of dNTPs, to form a duplex DNA which can then be ligated to the vector.
  • an appropriate enzyme such as a DNA polymerase in the presence of dNTPs
  • DNA encoding the second enzymatic RNA molecule is incorporated into the vector.
  • Suitable restriction endonuclease sites may also be provided to ease the construction of such a vector in DNA vectors or in requisite DNA vectors of an RNA expression system.
  • an expression vector useful in conjunction with ribozymes or deoxyribozym.es includes a bacterial, viral or eukaryotic promoter within a plasmid, cosmid, phagemid, virus, viroid, or phage vector.
  • Other suitable vectors include double-stranded DNA (dsDNA), partially double-stranded DNA, dsRNA, partially dsRNA, or single-stranded RNA (ssR A) or DNA (ssDNA). It should also be appreciated that useful vectors according to the present invention need not be circular.
  • an enzymatic DNA molecule-encoding nucleotide sequence is transcriptionally linked to a promoter sequence.
  • a vector according to the present invention may comprise an enzymatic RNA molecule under the control of a viral promoter, such as an Epstein-Barr Viras (EBV) promoter.
  • EBV Epstein-Barr Viras
  • a variety of viral promoters useful for this purpose are known in the art; see, e.g., those described in published PCT Application No. WO 93/23569, the disclosures of which are incorporated by reference herein.
  • one or more additional enzymatic RNA molecule-encoding nucleotide sequences are also included in the vector; said additional enzymatic RNA molecule-encoding sequences may be located on either side, or both sides, of a nucleotide sequence encoding the first enzymatic RNA molecule. Preferably, there are intervening nucleotides or nucleotide sequences between successive enzymatic RNA molecule-encoding sequences.
  • cellular splicing mechanisms within the target cell(s) may be utilized or integrated to cleave out the therapeutic second enzymatic RNA molecule(s) by encoding recognition sequences for the second enzymatic RNA molecules within the flanking sequences of the expressed transcript.
  • Multiple copies of the releasing first enzymatic RNA molecule may be provided to enhance release of the second (i.e. therapeutic) enzymatic RNA molecule if the turnover rate is slower than the degradation rate of the second enzymatic RNA molecule.
  • Intrabodies are single-chain antibodies derived from a parent monoclonal antibody in which the variable domains of the light and heavy chains are joined together by a flexible peptide linker. The resulting recombinant gene product retains the ability of the parent antibody to bind to and neutralize the target antigen.
  • intrabodies encompass monoclonals, single chain antibodies, V regions, and the like, as long as they bind to a target protein. The entire intrabody sequence can be encoded on an expression plasmid, and the plasmid can be transfected into cells, leading to intracellular expression of the intrabody protein and neutralization of its intracellular protein antigen. Intrabodies are reviewed and described in a variety of articles, including Marasco, W.A. (1997) Gene Ther. 4: 11-15 and Persic, L., et al (1997) Gene 187: 1-8.
  • Intracellular antibodies have found various applications, not only as research reagents but even more as therapeutic molecules. Initially, antibodies were microinjected into the cytoplasm of cells to block specifically the activity of cellular proteins. Recent advances in antibody engineering technology has led to two important developments: (1) the antigen binding domains of monoclonal antibodies can be expressed in recombinant form, e.g. as single-chain Fv fragments or Fab fragments; and (2) by using suitable expression systems, these fragments can be expressed in a variety of different cells, including mammalian cells.
  • recombinant antibodies can be directed to different subcellular compartments.
  • the attachment of a hydrophobic mammalian leader sequence to the N-terminus of the antibody fragment results in the secretion of the molecule to the extracellular environment.
  • Addition of an ER retention signal e.g. KDEL, SEQ ID NO: 4
  • an ER retention signal e.g. KDEL, SEQ ID NO: 4
  • the antibody fragment is directed to the plasma membrane, resulting in cell surface display.
  • antibody fragments can be directed to mitochondria by using a mitochondrial leader sequence, e.g.
  • Cytoplasmic expression is achieved by expression of the antibody fragments without any signal or leader sequences. These fragments can be imported into the nucleus by fusing a nuclear localization sequence, e.g. from the SV40 large T antigen (PKKKRKV) (SEQ ID NO: 5), to either the N- or C-terminus.
  • PKKKRKV SV40 large T antigen
  • intrabodies are capable of effectively neutralizing gpl20, the glycoprotein that studs the outer surface of HIV. Discussed in Marasco, W. et al, J Immunol Methods 231(l-2):223-38, 1999.
  • One such intrabody is sFvl05, a modified version of a human monoclonal antibody that recognizes the CD4 binding site of gpl20, which plays a critical role in the infection process. Stripped of its glycoprotein coat, the "naked" HIV is unable to infect new cells.
  • sFvl 05 inhibits the formation of syncytia, the lethal cell clusters that trigger widespread cell death.
  • intrabodies contemplated for use according to the present invention include those directed to both viral and cellular molecules involved in viral progression, replication, and related processes.
  • Genetic payloads delivered according to the invention include therapeutic nucleic acids that act by inhibiting a viral or cellular gene or polypeptide, as well as therapeutic molecules with direct therapeutic effects.
  • the invention includes genetic payloads comprising polynucleotides capable of expressing a polypeptide that promotes a host cell immune response to infection, for example.
  • Examples of such polypeptides include chemokines, immunomodulators, and cytokines, such as interferons, interleukins, tumor necrosis factor, and the like.
  • Cytokine immunotherapy is a modification of immunogene therapy and involves the administration of tumor cell vaccines that are genetically modified ex vivo or in vivo to express various cytokine genes.
  • cytokine gene transfer resulted in significant antitumor immune response (Fearon, et al (1990) Cell 60: 387-403; Wantanabe, et al (1989) Proc. Nat. Acad. Sci. USA, 86: 9456-9460).
  • the phages are used to deliver DNA encoding a cytokine, such as IL-12, IL-10, IL-2, GM-CSF, interferon-gamma (INF- ⁇ ), consensus interferon (IFN-Con-1), tumor necrosis factor (TNF), or an MHC gene, such as HLA-B7. Delivery of these genes will modulate the immune system, increasing the potential for host anti-infection immunity.
  • DNA encoding costimulatory molecules such as B7.1 and B7.2, ligands for CD28 and CTLA-4 respectively, can also be delivered to enhance T cell mediated immunity.
  • These genes can be co-delivered with cytokine genes, using the same or different promoters and optionally with an internal ribosome binding site.
  • Preferred therapeutic polypeptides of the invention include the interferons.
  • alpha and beta interferons are cytokines secreted by viras infected cells, which bind specific receptors on adjacent cells and protect them from infection.
  • the alpha and beta interferons form part of the immediate protective host response to invasion by viruses.
  • alpha and beta interferon also enhance the expression of class I and class II MHC molecules on the surface of infected cells, in this way, enhancing the presentation of viral antigens to specific immune cells.
  • Their presence can be demonstrated in body fluids during the acute phase of viras infection, according to methods known in the art.
  • Recombinant alpha and beta interferons are available and have been used for the treatment of chronic hepatitis B and C virus infections.
  • Gamma interferon is a cytokine secreted by TH1 CD4 cells. Its function includes enhancing specific T cell mediated immune responses.
  • the gamma interferon gene and polynucleotide sequence are available.
  • Certain viruses including vaccinia, show considerable resistance to the antiviral effects of interferons.
  • One of the early genes of vaccinia vims, K3L encodes a protein homologous to eIF-2a, which inhibits the action of PKR.
  • the E3L protein also binds dsRNA and inhibits PKR activation.
  • other poxviras- encoded proteins interfere with the actions of complement, IL-1 and TNFs. Proteins that inhibit interferon action are, therefore, targets for disruption by therapeutic nucleic acid molecules of the invention.
  • delivery vehicles may deliver multiple nucleic acids, such as, for example, both a gene encoding an interferon and a gene encoding an inhibitory molecule (e.g. antisense, intrabody, or ribozyme) directed against a viral protein that confers resistance to an interferon.
  • inhibitory molecule e.g. antisense, intrabody, or ribozyme
  • treatment means any manner in which the symptoms of a condition, infection, disorder or disease are ameliorated or otherwise beneficially altered. Treatment also encompasses any pharmaceutical use of the compositions herein.
  • amelioration of the symptoms of a particular disorder refers to any lessening, whether permanent or temporary, lasting or transient, that can be attributed to or associated with administration of the composition.
  • the ligand displaying genetic packages of the present invention can be delivered in vivo by administration to an individual, typically via systemic administration (e.g., oral, intravenous, intraperitoneal, intramuscular, subdermal, or infracranial fusion) or topical application (e.g., dermis and mucosal membranes).
  • systemic administration e.g., oral, intravenous, intraperitoneal, intramuscular, subdermal, or infracranial fusion
  • topical application e.g., dermis and mucosal membranes.
  • the bacteriophage of the present invention are capable of targeting a specific cell type for transduction.
  • Examples of specific cell types that may be targeted according to the invention include, but are not limited to, endothelial cells, fibroblasts, lymphocytes, neuronal cells, macrophages, keratinocytes, lung, vaginal, intestinal, stomach, trachea, nasal, kidney, ovarian, and testes epithelial cells, myocytes, myoblasts, hematopoietic cells, bone marrow cells, and stem cells.
  • the ligand displaying genetic packages of the present invention can be delivered to cells ex vivo.
  • Ex vivo treatment envisions withdrawal or removal of a population of cells from a patient, transduction with bacteriophages containing therapeutic nucleic acids, and re-implantation into the body of a patient. Exemplary procedures for performing generalized ex vivo gene therapy are described in U.S. Patent Nos. 5,399,346 and 5,665,350.
  • cells that have been transduced with phages ex vivo may be reintroduced into the patient or animal as described by a variety of methodologies. See, e.g., U.S. PatentNo.5,399,346, 5,665,350, PCT Publication No. WO 92/15676 (claiming priority to U.S. SerialNo. 667,169), PCT Publication No. WO 90/06997 (claiming priority to U.S. Serial No. 283,586).
  • Exemplary cell populations for ex vivo transduction, include any cell infected or vulnerable to infection, such as bone marrow cells, liver cells, pancreatic cells, all hemopoetic cells, and umbilical blood and cells.
  • Such cells may be processed to purify the desired cell population for transduction, for example to obtain a specified subset of cells for transduction.
  • Preferred methods of purification include various cell sorting techniques, such as antibody panning, FACS, and affinity chromatography using a matrix coupled to antibodies specifically reactive with the desired subset of cells. Isolated cells may then be transduced and re-introduced to a patient or, alternatively, the cells may be expanded in culture prior to re-introduction.
  • compositions, and complexes suitable for administration of the compounds, conjugates, compositions, and complexes provided herein include any such carriers known to those skilled in the art to be suitable for the particular mode of administration.
  • non-target vectors may be formulated as the sole pharmaceutically active ingredient in the composition or may be combined with other active ingredients.
  • epitopes and ligands may be formulated as the sole pharmaceutically active ingredient in the composition or may be combined with other active ingredients.
  • the non-target vectors can be administered by any appropriate route, for example, orally, parenterally, including intravenously, intradermally, subcutaneously, or topically, in liquid, semi-liquid or solid form and are formulated in a manner suitable for each route of administration. Preferred modes of administration depend upon the indication treated.
  • the non-target vectors may be formulated into pharmaceutical compositions suitable for local, intravenous and systemic application. Time release formulations are also desirable. Effective concentrations of the non-target vector are mixed with a suitable pharmaceutical carrier or vehicle.
  • an "effective amount" of a compound for treating a particular infection or associated disease is an amount that is sufficient to ameliorate, or in some manner reduce the microbial or viral load or symptoms associated with the disease.
  • Such amount may be administered as a single dosage or may be administered according to a regimen, whereby it is effective.
  • the amount may ameliorate infection or cure the disease but, typically, is administered in order to slow the rate of infection or associated disease progression. Repeated administration may be required to achieve the desired amelioration of infection and associated disease symptoms.
  • the non-target vectors of the present invention are particularly well-suited for oral administration and possess the ability to deliver therapeutic gene sequences on a systemic level, unlike most of the gene delivery vectors presently in testing and in use.
  • certain non-mammalian viruses such as phage
  • phage are able to withstand harsh environments and conditions such as those common to the mammalian digestive tract; thus, they are ideally suited for oral/systemic formulations and administration.
  • Protease-resistant ligands are particularly preferred for use in formulations and compositions designed for oral/systemic administration.
  • Therapeutically effective concentrations and amounts may be determined empirically by testing the conjugates and complexes in known in vitro and in vivo systems, such as those described here; dosages for humans or other animals may then be extrapolated therefrom.
  • the non-target vector is included in the pharmaceutically acceptable carrier in an amount sufficient to exert a therapeutically useful effect in the absence of undesirable side effects on the patient treated.
  • the non-target vectors may be delivered as pharmaceutically acceptable salts, esters or other derivatives of the conjugates include any salts, esters or derivatives that may be readily prepared by those of skill in this art using known methods for such derivatization and that produce compounds that may be administered to animals or humans without substantial toxic effects. It is understood that number and degree of side effects depends upon the condition for which the conjugates and complexes are administered. For example, certain toxic and undesirable side effects are tolerated when treating life-threatening infections, but would not be tolerated when treating infections of lesser consequence.
  • vaccines and pharmaceutical compositions are provided for the prevention of infection, and complications thereof.
  • the vaccines are used to prevent infection of mammals.
  • the pharmaceutical compositions generally comprise one or more peptides or polypeptides, containing one or more epitopes or ligands capable of binding a cell surface receptor associated with infectious agent binding or intemalization, and a physiologically acceptable carrier.
  • the epitope or ligand was identified according to methods of the invention.
  • the vaccines comprise one or more of the above peptides or polypeptides and an adjuvant, for enhancement of the immune response.
  • the pharmaceutical compositions when used as vaccines, may be administered by injection (e.g., intramuscular, intravenous or subcutaneous), intranasally (e.g., by aspiration) or orally. Between 1 and 4 doses may be administered for a 2-6 week period. Preferably, two doses are administered, with the second dose 2-4 weeks later than the first.
  • a suitable dose is an amount of polypeptide that is effective to raise antibodies in a treated mammal that are sufficient to protect the mammal from infection for a period of time.
  • the amount of polypeptide present in a dose ranges from about 1 pg to about 100 mg per kg of host, typically from about 10 pg to about 1 mg, and preferably from about 100 pg to about 1 ⁇ g. Suitable dose sizes will vary with the size of the animal, but will typically range from about 0.01 mL to about 5 mL for 10-60 kg animal. While any suitable carrier known to those of ordinary skill in the art may be employed in the pharmaceutical compositions of this invention, the type of carrier will vary depending on the mode of administration. For parenteral administration, such as subcutaneous injection, the carrier preferably comprises water, saline, alcohol, a fat, a wax or a buffer.
  • any of the above carriers or a solid carrier such as mannitol, lactose, starch, magnesium stearate, sodium saccharine, talcum, cellulose, glucose, sucrose, and magnesium carbonate, may be employed.
  • Biodegradable microspheres e.g., polylactic galactide
  • adjuvants may be employed in the vaccines of this invention to nonspecifically enhance the immune response.
  • Most adjuvants contain a substance designed to protect the antigen from rapid catabolism, such as aluminum hydroxide or mineral oil, and a nonspecific stimulator of immune response, such as lipid A, Bordella pertussis or Mycobacterium tuberculosis.
  • lipid A lipid A
  • Bordella pertussis or Mycobacterium tuberculosis lipid A
  • Such adjuvants are commercially available as, for example, Freund's Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit, MI) and Merck Adjuvant 65 (Merck and Company, Inc., Rahway, NJ).
  • Solutions or suspensions used for parenteral, intradermal, subcutaneous, or topical application of any composition or compound of the invention can include any of the following components: a sterile diluent, such as water for injection, saline solution, fixed oil, polyethylene glycol, glycerine, propylene glycol or other synthetic solvent; antimicrobial agents, such as benzyl alcohol and methyl parabens; antioxidants, such as ascorbic acid and sodium bisulfite; chelating agents, such as ethylenediaminetetraacetic acid (EDTA); buffers, such as acetates, citrates and phosphates; and agents for the adjustment of toxicity such as sodium chloride or dextrose.
  • a sterile diluent such as water for injection, saline solution, fixed oil, polyethylene glycol, glycerine, propylene glycol or other synthetic solvent
  • antimicrobial agents such as benzyl alcohol and methyl parabens
  • antioxidants such as as
  • suitable carriers include physiological saline or phosphate buffered saline (PBS), and solutions containing thickening and solubilizing agents, such as glucose, polyethylene glycol, and polypropylene glycol and mixtures thereof.
  • PBS physiological saline or phosphate buffered saline
  • suitable carriers include physiological saline or phosphate buffered saline (PBS), and solutions containing thickening and solubilizing agents, such as glucose, polyethylene glycol, and polypropylene glycol and mixtures thereof.
  • Liposomal suspensions may also be suitable as pharmaceutically acceptable carriers. These may be prepared according to methods known to those skilled in the art.
  • the non-target vector, peptides, and polypeptides can also be mixed with other active materials that do not impair the desired action or with materials that supplement the desired action.
  • the non-target vectors may be prepared with carriers that protect them against rapid elimination from the body, such as time release formulations or coatings.
  • Such carriers include controlled release formulations, such as, but not limited to, implants and microencapsulated delivery systems, and biodegradable, biocompatible polymers, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, polyorthoesters, polylactic acid and others.
  • the composition may be applied during surgery using a sponge, such as a commercially available surgical sponges (see, e.g., U.S. Patent Nos. 3,956,044 and 4,045,238) that has been soaked in the phage.
  • the vectors e.g.
  • phage may also be applied in pellets (such as Elvax pellets (ethylene-vinyl acetate copolymer resin).
  • Preferential carriers in this regard include a gene-activated matrix (GAM), as described in PCT Publication No. WO 97/38729 (claiming priority to U. S. Serial No. 08/631,334) and PCT Publication No. WO 95/22611 (claiming priority to U. S. Serial No. 08/199,780, and 08/316,650).
  • GAM gene-activated matrix
  • a gene-activated matrix is any matrix material containing DNA encoding a therapeutic agent, for example, non-target vectors of this invention. Further, such gene-activated matrices are preferentially derived from any biocompatible material.
  • the vector or peptide/polypeptide should be provided in a composition that protects it from the acidic environment of the stomach, for example in an enteric coating or in combination with an antacid.
  • bacteria that are "infected" with the phage as the vector may be used for oral administration purposes, as they have the capacity to propagate in the intestine and release the phage.
  • Oral compositions will generally include an inert diluent or an edible carrier and may be compressed into tablets or enclosed in gelatin capsules.
  • Tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder, such as microcrystalline cellulose, gum tragacanth and gelatin; an excipient such as starch and lactose, a disintegrating agent such as, but not limited to, alginic acid and corn starch; a lubricant such as, but not limited to, magnesium stearate; a glidant, such as, but not limited to, colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; and a flavoring agent such as peppermint, methyl salicylate, and fruit flavoring.
  • a binder such as microcrystalline cellulose, gum tragacanth and gelatin
  • an excipient such as starch and lactose, a disintegrating agent such as, but not limited to, alginic acid and corn starch
  • a lubricant such as, but not limited to, magnesium stearate
  • the compounds may be packaged as articles of manufacture containing packaging material or a container, one or more conjugates and complexes or compositions as provided herein within the packaging material, and a label that indicates the indication for which the conjugate is provided and/or instmctions for use of the compound.
  • the nucleic acid delivery vehicles may be assessed in any number of in vitro and in vivo model systems.
  • the vector should bind to a cell in a specific manner and internalize (likely through endosomal. pathway).
  • the transgene should be expressed by the host (targeted) cells.
  • appropriate assays include, without limitation, a binding assay to verify that the vectors attach to target cell, a displacement assay to show that the ligand-bearing constructs can be displaced by excess free ligand, an intemalization assay to demonstrate that vectors are internalized and a functional assay to show that transgene is active in host cell (See e.g., U.S. Serial No. 09/141,631, corresponding to U.S. Provisional Serial No. 60/057,067).
  • the present invention provides platform technology consisting of ligand displaying genetic packages and phage particles useful for combating a variety of intracellular infections, particularly infections with infectious agents associated with high lethality and incapacity.
  • the invention includes compounds and compositions that may be used to deliver a genetic payload to a cell that is infected or at risk of infection by an infectious agent.
  • the invention is used to treat patients suffering from or at risk of a disease caused by infection with a potential biowarfare agent.
  • Patients include any infectable animal or plant, including humans, domestic animals, livestock, fish, birds, crops and other plants with commercial value, for example.
  • human infectious diseases include, but are not limited to, Legionnaire's disease, Epstein- Barr, rickettsia, brucellosis, Johne's disease, cholera, glanders, pneumatic plague, Q fever, smallpox, Venezuelan equine encephalitis, viral hemorrhagic fevers, malaria, tuberculosis, AIDS, and hepatitis C.
  • the invention provides methods for delivering ligand displaying genetic packages to cells, as well as methods of treating or preventing infection.
  • the invention provides methods of treating a patient infected with a potentially lethal viras comprising delivering a therapeutic gene to infected cells via the same receptor/moiety utilized by the virus for entry.
  • the invention utilizes vectors or delivery vehicles containing ligands capable of binding to such cell receptors/moieties.
  • delivery vehicles are non-mammalian viruses, for reasons set forth above. In particular embodiments, these non-mammalian vimses are filamentous phage.
  • the invention provides a method for delivery of an antimicrobial agent that includes contacting a cell with a non-mammalian virus present a ligand on it surface, the ligand being specific for a protein present on the surface of a mammalian cell.
  • the virus itself comprises a heterologous nucleic acid sequence that encodes an anti-viral agent.
  • the invention provides a similar method for delivery of an agent selected to counteract infection by a non-viral microbe. In this method, the heterologous nucleic acid sequence provided by the method encodes an agent effective against the non- viral microbe.
  • the invention provides a method for treating or slowing a viral infection that includes contacting a cell with a non-mammalian vims presenting a ligand on its surface.
  • the ligand is specific for a protein present in the cell surface of a mammalian cell. This protein is preferably a viral receptor or a protein upregulated upon viral infection.
  • the non-mammalian virus harbors a heterologous nucleic acid sequence comprising an anti- viral agent.
  • Methods of the infection directed to treating infection by vimses or other receptor-mediated intracellular microbes can achieve this result through a variety of mechanisms, as described above.
  • Different therapeutic nucleic acids may employ different mechanisms of fighting infection. Accordingly, several therapeutic nucleic acids may be delivered according to methods of the invention. These different therapeutic nucleic acids may act through the same or different mechanisms.
  • therapeutic genes and methods of the invention may combat infection by mechanisms including the destruction of infected cells and halting or slowing viral replication. In certain embodiments, the methods slow viral replication sufficiently to allow the host's response to ward off infection.
  • the invention also provides methods of identifying and selecting epitopes and ligands of infectious agents that are involved in cell binding and intemalization (i. e. portal of entry ligands) and their use as vaccines or as competitive inhibitors of entry. Accordingly, the invention includes ligand displaying genetic packages that display a ligand identified according to such methods and methods of combating infection by delivering such ligand displaying genetic packages to a patient.
  • the invention provides methods of eliciting a host immune response directed against an intracellular infectious agent, methods of vaccination to prevent infection by an intracellular infectious agent, and a vaccine.
  • portal of entry ligands including those identified according to methods of the invention, are used as vaccines to elicit a protective immune response in a patient infected or at risk of infection.
  • the use of portal of entry ligands offers advantages over the traditional use of immunodominant epitopes, including those associated with viral coat proteins, as vaccines. Since portal of entry ligands contribute to defining the tropism of the infectious agent and its associated disease, such ligands are more likely to be conserved and less likely to undergo changes, such as, for example, amino acid sequence variations through evolution or mutation.
  • portal of entry ligands and epitopes useful as vaccines include any such ligand or epitope capable of eliciting an immune response. These include naturally-occurring ligands and epitopes derived therefrom, as well as any epitope identified using LIVETM methods, as described above. Accordingly, the invention provides methods of eliciting an immune response against an intracellular infectious agent and vaccinating a patient by delivering a portal of entry ligand or epitope to the patient in an amount sufficient to elicit an immune response or a protective immune response.
  • a ligand or epitope to elicit an immune response can be tested according to any method available in the art, including, for example, by immunizating an appropriate animal model with a ligand or epitope and subsequently testing (e.g. by western blot) to determine if antibodies directed against the ligand or epitope are generated by the immunized animal.
  • the invention provides methods of identifying a viral epitope or ligand that binds to and/or internalizes into cells capable of being infected by a viras.
  • a phage display library comprising cDNAs from a viras fused to a phage coat protein is screened according to LIVETM methods using cells capable of being infected by the viras.
  • the phage further comprise nucleic acids that express a detectable/selectable product (e.g.
  • nucleic acid or polypeptide that is expressed in a cell that binds and/or internalizes the phage, thereby permitting the detection and/or selection of cells containing phage and, ultimately, the identification of the epitope or ligand expressed by the viral cDNA.
  • a viral epitope or ligand that binds to and/or internalizes into cells capable of being infected by a viras is identified by screening a phage display library comprising random peptides according to LIVETM methods using cells capable of being infected by the virus.
  • viral epitopes and ligands to cell surface receptors associated with infection are identified by infecting cells with an infectious agent and subsequently screening according to LIVETM methods for ligands that are internalized by the infected cells. Screening using LIVETM methods, as described above, offers advantages over other phage display screening methods.
  • the criteria for apositive "hit" using LINETM methods is that the phage must be able to bind, be internalized, and enable detection of the internalized ligand by detecting a marker, such as, for example, by expressing a phage vector genomic D ⁇ A containing a reporter/selectable gene in the target cell or allowing direct nucleic acid detection (e.g.
  • the method provided by the invention offers advantages associated with their ability to simultaneously select for intemalization and expression, thereby allowing the identification of portal-specific epitopes and ligands.
  • Identified portal-specific epitopes and ligands are used according to the invention to deliver nucleic acids to infected cells or as antigens for immunization.
  • EGF receptor targeted phagemid particles are prepared from an appropriate host bacteria (XLl-blue; Statagene) that has been transformed with a modified pUC250- EGF phagemid (Larocca, D. et al. Molecular Therapy 3:476-84, 2001).
  • the phagemid contains mouse EGF in place of human EGF and has an anti-sense version of the mousepox p28 protein under the transcriptional control of a CMV (cytomegeloviras) immediate early gene promoter/enhancer.
  • CMV cytomegeloviras
  • the zinc finger motif containing p28 protein has been shown to be an important determinant of poxviras pathogenicity (Senkevich, T.G. et al.
  • a susceptible strain of mouse (6-8 week old female), Balb/c, is infected with ECTN-602 mouse pox vims by footpad inoculation with 10 3 pfu of viras.
  • Mousepox causes an acute systemic infection in this strain with high viral titers in liver and spleen and high mortality at two weeks following inoculation.
  • mice At the end of two weeks, the surviving mice are sacrificed and viral titers obtained from liver and spleen. An increase in survival of ECTV-602 inoculated mice treated with EGF targeted/p28 anti-sense phagemid particles indicates a positive anti-viral effect of the phagemid treatment.

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Abstract

L'invention concerne une technologie plate-forme pour le traitement d'infections intracellulaires. L'invention concerne des compositions et des procédés comportant des vecteurs sans cible spécifique qui visent des cellules susceptibles de s'infecter par le biais de ligands liés qui se lient et s'internalisent par l'intermédiaire de récepteurs/fractions de surface cellulaire associés à l'infection. Les vecteurs comprennent des séquences d'acides nucléiques exogènes qui s'expriment dès l'internalisation dans une cellule cible. Les ligands associés aux vecteurs et les molécules d'acides nucléiques peuvent être altérés de manière à cibler différents agents infectieux. En outre, l'invention concerne des procédés d'identification d'épitopes et de ligands pouvant diriger l'internalisation d'un vecteur et verrouiller l'entrée virale.
PCT/US2003/010081 2002-04-05 2003-04-01 Compositions et procedes pour administration genique a specificite de porte et traitement des infections Ceased WO2003086276A2 (fr)

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WO2006095345A3 (fr) * 2005-03-08 2007-05-24 Univ Ramot Bacteriophages transportant des medicaments cibles
US20110129525A1 (en) * 2008-02-20 2011-06-02 Universiteit Gent Mucosal membrane receptor and uses thereof
WO2016077526A1 (fr) 2014-11-12 2016-05-19 Siamab Therapeutics, Inc. Composés interagissant avec le glycane et procédés d'utilisation
WO2017083582A1 (fr) 2015-11-12 2017-05-18 Siamab Therapeutics, Inc. Composés interagissant avec le glycane et méthodes d'utilisation
WO2017079314A3 (fr) * 2015-11-02 2017-06-22 Singh Biotechnology, Llc Anticorps à domaine unique dirigés contre des antigènes intracellulaires
US9850321B2 (en) 2014-10-23 2017-12-26 Singh Molecular Medicine, Llc Single domain antibodies directed against intracellular antigens
US9879087B2 (en) 2014-11-12 2018-01-30 Siamab Therapeutics, Inc. Glycan-interacting compounds and methods of use
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US11401330B2 (en) 2016-11-17 2022-08-02 Seagen Inc. Glycan-interacting compounds and methods of use

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US5736388A (en) * 1994-12-30 1998-04-07 Chada; Sunil Bacteriophage-mediated gene transfer systems capable of transfecting eukaryotic cells
US6054312A (en) * 1997-08-29 2000-04-25 Selective Genetics, Inc. Receptor-mediated gene delivery using bacteriophage vectors
US6451527B1 (en) * 1997-08-29 2002-09-17 Selective Genetics, Inc. Methods using genetic package display for selecting internalizing ligands for gene delivery

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US7985573B2 (en) 2005-03-08 2011-07-26 Ramot At Tel-Aviv University Ltd. Targeted drug-carrying bacteriophages
WO2006095345A3 (fr) * 2005-03-08 2007-05-24 Univ Ramot Bacteriophages transportant des medicaments cibles
US20110129525A1 (en) * 2008-02-20 2011-06-02 Universiteit Gent Mucosal membrane receptor and uses thereof
US9052317B2 (en) * 2008-02-20 2015-06-09 Universiteit Gent Mucosal membrane receptor and uses thereof
US9636417B2 (en) 2008-02-20 2017-05-02 Universiteit Gent Mucosal membrane receptor and uses thereof
US9850321B2 (en) 2014-10-23 2017-12-26 Singh Molecular Medicine, Llc Single domain antibodies directed against intracellular antigens
WO2016077526A1 (fr) 2014-11-12 2016-05-19 Siamab Therapeutics, Inc. Composés interagissant avec le glycane et procédés d'utilisation
US9879087B2 (en) 2014-11-12 2018-01-30 Siamab Therapeutics, Inc. Glycan-interacting compounds and methods of use
USRE49435E1 (en) 2014-11-12 2023-02-28 Seagen Inc. Glycan-interacting compounds and methods of use
EP4183806A2 (fr) 2014-11-12 2023-05-24 Seagen Inc. Composés interagissant avec le glycane et procédés d'utilisation
WO2017079314A3 (fr) * 2015-11-02 2017-06-22 Singh Biotechnology, Llc Anticorps à domaine unique dirigés contre des antigènes intracellulaires
IL258742A (en) * 2015-11-02 2018-06-28 Singh Biotechnology Llc Single domain antibodies directed against intracellular antigens
WO2017083582A1 (fr) 2015-11-12 2017-05-18 Siamab Therapeutics, Inc. Composés interagissant avec le glycane et méthodes d'utilisation
US11028181B2 (en) 2015-11-12 2021-06-08 Seagen Inc. Glycan-interacting compounds and methods of use
US11401330B2 (en) 2016-11-17 2022-08-02 Seagen Inc. Glycan-interacting compounds and methods of use
US11253609B2 (en) 2017-03-03 2022-02-22 Seagen Inc. Glycan-interacting compounds and methods of use

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